Chemical Compounds

ABSTRACT

The invention concerns compounds of Formula (I) 
     
       
         
         
             
             
         
       
     
     or pharmaceutically-acceptable salts thereof, wherein R 1  and R 2  have any of the meanings defined hereinbefore in the description; processes for their preparation, pharmaceutical compositions containing them and their use in the treatment of cell proliferative disorders.

This application is a continuation of U.S. patent application Ser. No.14/160,650, filed Jan. 22, 2014, issuing, which claims the benefit under35 U.S.C. §119(a)-(d) of Patent Application No. 13305078.1 (EP), filedJan. 23, 2013.

The invention concerns certain novel aminopyrazine derivatives, orpharmaceutically-acceptable salts thereof, which possess anti-canceractivity and are accordingly useful in methods of treatment of the humanor animal body. The invention also concerns processes for themanufacture of said aminopyrazine derivatives, pharmaceuticalcompositions containing them and their use in therapeutic methods, forexample in the manufacture of medicaments for use in the prevention ortreatment of cancers in a warm-blooded animal such as man, including usein the prevention or treatment of cancer.

The present invention also relates to aminopyrazine derivatives that areselective inhibitors of the PI3-kinase family of enzymes (which isalternatively known as the phosphatidylinositol-3-kinase family or PI3Kfamily), particularly of PI3K-α and PI3K-δ isoforms, and are, forexample, useful for anti-tumour therapy.

In the area of cancer it has in recent years been discovered that a cellmay become cancerous by virtue of the transformation of a portion of itsDNA into an oncogene, that is a gene which, on activation, leads to theformation of malignant tumour cells (Bradshaw, Mutagenesis, 1986, 1,91). Several such oncogenes give rise to the production of peptides,which are Kinases, a class of enzymes that are capable ofphosphorylating their protein or lipid substrates. There are severalclasses of kinases.

Firstly, tyrosine kinases, which may be receptor tyrosine kinases or nonreceptor tyrosine kinases. Various classes of receptor tyrosine kinasesare known (Wilks, Advances in Cancer Research, 1993, 60, 43-73) based onfamilies of growth factors, that can bind to the extracellular surfaceof different receptor tyrosine kinases; as an example the classificationincludes Class I receptor tyrosine kinases comprising the EGF family ofreceptor tyrosine kinases. Non-receptor tyrosine kinases are locatedintracellularly; various classes of non-receptor tyrosine kinases areknown including the Src family such as the Src, Lyn, Fyn and Yestyrosine kinases.

Secondly, certain kinases belong to the class of serine/threoninekinases which are also located intracellularly. Serine/threonine kinasesignalling pathways include the Raf-MEK-ERK cascade and those downstreamof PI3-kinase such as PDK-1, AKT and mTOR (Blume-Jensen and Hunter,Nature, 2001, 411, 355).

It is also known that certain other kinases belong to the class of lipidkinases, which are located intracellularly and are, as for the abovementioned kinases, involved in the transmission of biochemical signalssuch as those that influence tumour cell growth and invasiveness.Various classes of lipid kinases are known including the aforementionedPI3-kinase family.

It is now well understood that deregulation of oncogenes andtumour-suppressor genes contributes to the formation of malignanttumours, for example by way of increased cell proliferation or increasedcell survival. It is also now known that signalling pathways mediated bythe PI3-kinase family have a central role in a number of cell processesincluding proliferation and survival, and deregulation of these pathwaysis a causative factor across a wide spectrum of human cancers and otherdiseases (Katso et al., Annual Rev. Cell Dev. Biol., 2001, 17: 615-617and Foster et al., J. Cell Science, 2003, 116: 3037-3040).

The PI3-kinase family of lipid kinases is a group of enzymes thatphosphorylate the 3-position of the inositol ring ofphosphatidylinositol (PI). Three major groups of PI3-kinase enzymes areknown which are classified according to their physiological substratespecificity (Vanhaesebroeck et al., Trends in Biol. Sci., 1997, 22, 267;Engleman et al., Nature Review Genetics, 2006, 7, 607). Class IIIPI3-kinase enzymes phosphorylate PI alone. In contrast, Class IIPI3-kinase enzymes phosphorylate both PI and PI4-phosphate [abbreviatedhereinafter to PI(4)P]. Class I PI3-kinase enzymes phosphorylate PI,PI(4)P and PI4,5-bisphosphate [abbreviated hereinafter to PI(4,5)P2],although only PI(4,5)P2 is believed to be the physiological cellularsubstrate. Phosphorylation of PI(4,5)P2 produces the lipid secondmessenger PI3,4,5-triphosphate [abbreviated hereinafter to PI(3,4,5)P3].More distantly related members of this superfamily are Class IV kinasessuch as mTOR and DNA-dependent protein kinase that phosphorylateserine/threonine residues within protein substrates. The most studiedand understood of these lipid kinases are the Class I PI3-kinaseenzymes.

Class I PI3-kinases are heterodimers consisting of a p110 catalyticsubunit and a regulatory subunit, and the family is further divided intoClass Ia and Class Ib enzymes on the basis of regulatory partners andmechanism of regulation (Engleman et al., Nature Review Genetics, 2006,7, 607). Class Ia enzymes consist of three distinct catalytic subunits(p110α, p110β and p110δ, by nomenclature define the PI3-Kinase isoformas α, β or δ respectively) that dimerise with five distinct regulatorysubunits (p85α, p55α, p50α, p85β and p55γ), with all catalytic subunitsbeing able to interact with all regulatory subunits to form a variety ofheterodimers. Class Ia PI3-kinase enzymes are generally activated inresponse to growth factor-stimulation of receptor tyrosine kinases, viainteraction of the regulatory subunit SH2 domains with specificphospho-tyrosine residues of the activated receptor or adaptor proteinssuch as IRS-1. Both p110α and p110β are widely expressed across celltypes and tissues, whereas p110δ expression is more restricted toleukocyte populations and some epithelial cells. In contrast, the singleClass Ib enzyme consists of a p110γ catalytic subunit that interactswith a p101 regulatory subunit. Furthermore, the Class Ib enzyme isactivated in response to G-protein coupled receptor (GPCR) systems aswell as by the mechanisms described above.

There is now considerable evidence indicating that Class Ia PI3-kinaseenzymes, contribute to tumourigenesis in a wide variety of humancancers, either directly or indirectly (Vivanco and Sawyers, NatureReviews Cancer, 2002, 2, 489-501). In particular, the PIK3CA gene whichencodes the p110α catalytic subunit of PI3-kinase is widely implicatedin tumourigenesis. Activating point mutations, most frequently found inthe helical or catalytic domains of p110α, increase the PI3-kinaseactivity of the holoenzyme and can transform cells. They have beenreported, particularly, as somatically occurring mutations atsignificant frequencies across a wide range of tumour types (Samuels etal., Science, 2004, 304, 554; Samuels et al., Cancer Cell, 2005, 7, 561;Engleman et al., Nature Review Genetics, 2006, 7, 607; Zhao L and Vogt PK, Oncogene 2008, 27 5486). Tumour-related mutations in p85α have alsobeen identified in cancers such as those of the ovary and colon (Philpet al., Cancer Research, 2001, 61, 7426-7429). Furthermore, the p110αsubunit is amplified in some tumours such as those of the ovary(Shayesteh et al., Nature Genetics, 1999, 21, 99-102) and cervix (Ma etal., Oncogene, 2000, 19, 2739-2744).

In addition to direct effects, it is believed that activation of ClassIa PI-3 kinase contributes to tumourigenic events that occur upstream insignalling pathways, for example by way of ligand-dependent orligand-independent activation of receptor tyrosine kinases, GPCR systemsor integrins (Vara et al., Cancer Treatment Reviews, 2004, 30, 193-204).Examples of such upstream signalling pathways include over-expression ofthe receptor tyrosine kinase Erb2 in a variety of tumours leading toactivation of PI 3-kinase-mediated pathways (Harari et al., Oncogene,2000, 19, 6102-6114) and over-expression of the oncogene Ras(Kauffmann-Zeh et al., Nature, 1997, 385, 544-548). In addition, ClassIa PI3-kinases may contribute to tumourigenesis caused by variousdownstream signalling events. For example, loss of the effect of thePTEN tumour-suppressor phosphatase that catalyses conversion ofPI(3,4,5)P3 back to PI(4,5)P2 is associated with a very broad range oftumours via deregulation of PI3-kinase-mediated production ofPI(3,4,5)P3 (Simpson and Parsons, Exp. Cell Res., 2001, 264, 29-41).Furthermore, augmentation of the effects of other PI3-kinase-mediatedsignalling events is believed to contribute to a variety of cancers, forexample by activation of Akt (Nicholson and Anderson, CellularSignalling, 2002, 14, 381-395).

Hence the common deregulation of PI3-kinase together with those ofupstream and downstream signalling pathways collectively make it one ofthe most commonly deregulated pathways in human cancer (Hennessey etal., Nature Reviews Drug Discovery, 2005, 4, 988).

In addition to a role in mediating proliferative and survival signallingin tumour cells, there is also good evidence that Class Ia PI3-kinaseenzymes will also contribute to tumourigenesis via its function intumour-associated stromal cells. For example, PI3-kinase signalling isknown to play an important role in mediating angiogenic events inendothelial cells in response to pro-angiogenic factors such as VEGF(Abid et al., Arterioscler. Thromb. Vasc. Biol., 2004, 24, 294-300). AsClass I PI3-kinase enzymes are also involved in motility and migration(Sawyer, Expert Opinion Investig. Drugs, 2004, 13, 1-19), PI3-kinaseinhibitors should provide therapeutic benefit via inhibition of tumourcell invasion and metastasis.

In addition, Class I PI3-kinase enzymes play an important role in theregulation of immune cells with PI3-kinase activity contributing topro-tumourigenic effects of inflammatory cells (Coussens and Werb,Nature, 2002, 420, 860-867). Indeed, the Class Ia PI3-kinase enzyme,PI3-kinase δ, is particularly implicated in tumourigenesis inhaematological malignancies, such as Chronic Lymphoctyic Leukaemia(CLL), Acute Lymphoblastic Leukaemia (ALL) and Mantle Cell Lymphoma(MCL). Elevated-signalling of PI3K (mainly p110δ) is reported in a widerange of malignant lymphoid cells (Herman et al., Blood 2010, 116. 2078;Ikeda et al., Blood, 2010, 116, 1460; Uddin et al., Blood, 2006, 108,4178; Rudelius et al., Blood 2006, 108, 1668; Garcia-Martinez., Br JCancer, 2011, 104, 1116; Renne et al., Leukemia, 2007, 2, 780). This hasled to the development of agents targeting PI3-kinase δ, with promisinginitial clinical results in haematological malignancies. (Castillo etal., Expert Opinion on Investigational Drugs, 2012, 21, 15).

These findings suggest that pharmacological inhibitors of Class IPI3-kinase enzymes should be of therapeutic value for treatment of thevarious forms of the disease of cancer comprising solid tumours such ascarcinomas and sarcomas and the leukaemias and lymphoid malignancies.

Early studies, both pre-clinical and clinical, exploring thephysiological and pathological roles of the PI3-kinase enzyme, havelargely used agents with limited kinase inhibition selectivity, eitherstretching across the wider kinase families, across the PI3-kinasefamily, or across the PI3-kinase Class 1 family. Hence, there is a needfor more selective pharmaceutical PI3-kinase Class 1 inhibitors toprovide useful therapeutic agents with potential to deliver an improvedtherapeutic margin over the initial agents that entered the clinic.

Generally, the compounds of the present invention possess potentinhibitory activity against a subset of Class I PI3-kinase enzymes,particularly against Class Ia PI3-kinase-α and -δ isoforms, withrelative sparing of the -γ and particularly the -β isoform. Thecompounds are also selective against wider PI3-kinase family and thewider kinome. Such compounds possess sufficient potency against Class IPI3-kinase enzymes that they may be used in an amount sufficient toinhibit a subset of Class IPI 3-kinase isoforms, particularly to inhibitClass Ia PI3-kinase enzymes-α and -δ, whilst demonstrating littleactivity against other kinases.

The understanding of the deregulation of PI3-kinase signalling in humancancer and other diseases offers the prospect of targeting a subset ofpatients most likely to benefit from treatment of the agents describedin this patent, through a process known as Personalised Healthcare (PHC)or Personalised Medicine. For these agents, patients whose diseasedepends on elevated or otherwise altered PI3K-α signalling and/or PI3K-δsignalling may particularly benefit from treatment. It is well known inthe art that diagnostics can be used to provide a response-predictionbiomarker readout. Such diagnostics could measure one or more readoutsof pathway deregulation such as, but not restricted to, mutation in thePIK3CA, PTEN or p85 (PIK3R) genes, amplification or increased copynumber of the PIK3CA gene, overexpression or elevated activity of thePI3K-α and/or -δ isoform, or use of a phosphobiomarker readout withinthe pathway such as phospho-RTK or phospho-AKT. In addition, themeasurement of mutation status or activation status of additional genes,such as Kras, a potential marker of resistance in tumours with aberrantor deregulated PIK3CA or PI3K-α (Engelman et al., Nature Medicine, 200814, p 1351-1355; Ihle et al., Cancer Research, 2009, 69, p 143-160;Janku et al., Molecular Cancer Therapeutics, 2011, 10, p 558-564), couldhelp increase the predictivity of a Personalised Medicine approach.Alternatively, in another targeted but less specific approach, thetreatment could be focused in disease subsets where the deregulation ofthe relevant PI3K isoforms is known to be most prevalent.

The compounds described could be used to target disease, either alone orin combination with another pharmaceutical agent or agents. CombiningPI3-kinase inhibitors with other therapies may improve efficacy byovercoming resistance mechanisms, either innate, or induced in responseto the PI3-kinase agent. There is substantial pre-clinical data tosupport such an approach (Courtney et al, J Clin Oncol, 2010, 28, 1075;Engleman et al., Nature Review Genetics, 2006, 7, 607). One approach is‘intra-pathway’ combinations with agents modulating other axes in thePI3-kinase signalling pathways (e.g. mTOR, AKT, RTK, other PI3-kinaseagent). A second approach is ‘inter-pathway’ combinations whereinhibition of more than one signalling pathway may be beneficial overinhibition of a single pathway (e.g. combined with MEK inhibitors, Rafinhibitors, Bcl family modulators, RTK inhibitors or DNA damagesignalling modulators such as PARP inhibitors). Other approaches includewhere the PI3-kinase inhibitor is combined with agents or regimens thatare already established in clinical practice, so called Standard of Care(SoC) approaches, or combinations with agents targeting non tumour cellmechanisms such as tumour stromal cell or via the immune system.

In addition to tumourigenesis, there is evidence that Class I PI3-kinaseenzymes play a role in other diseases (Wymann et al., Trends inPharmacological Science, 2003, 24, 366-376). Both Class Ia PI3-kinaseenzymes, particularly PI3K-δ, and the single Class Ib enzyme (PI3K-γ)have important roles in cells of the immune system (Koyasu, NatureImmunology, 2003, 4, 313-319) and thus they are therapeutic targets forinflammatory and allergic indications. Inhibition of PI3-kinase is also,as described earlier, useful to treat cardiovascular disease viaanti-inflammatory effects or directly by affecting cardiac myocytes(Prasad et al., Trends in Cardiovascular Medicine, 2003, 13, 206-212).Thus inhibitors of Class I PI3-kinase enzymes may be of value in theprevention and treatment of a wide variety of diseases in addition tocancer.

The compounds, i.e. the aminopyrazine derivatives, of the invention havebeen found to possess potent anti-tumour activity, being useful ininhibiting the uncontrolled cellular proliferation which arises frommalignant disease. Without wishing to imply that the compounds disclosedin the present invention possess pharmacological activity only by virtueof an effect on a single biological process, it is believed that thecompounds provide an anti-tumour effect by way of inhibition of Class IPI3-kinase enzymes, particularly by way of inhibition of a subset of theClass Ia PI3-kinase enzymes, more particularly by way of inhibition ofthe PI3K-α and -δ isoforms.

The compounds of the present invention may also be useful in inhibitingthe uncontrolled cellular proliferation which arises from variousnon-malignant diseases such as inflammatory diseases (for examplerheumatoid arthritis and inflammatory bowel disease), fibrotic diseases(for example hepatic cirrhosis and lung fibrosis), glomerulonephritis,multiple sclerosis, psoriasis, benign prostatic hypertrophy (BPH),hypersensitivity reactions of the skin, blood vessel diseases (forexample atherosclerosis and restenosis), allergic asthma,insulin-dependent diabetes, diabetic retinopathy and diabeticnephropathy.

Proline amides have been disclosed as selective PI3K-α selective agentsby Novartis in International Patent Applications WO2009/080705,WO2010/029082 and WO2011/000905. Aminopyrazine containing ATR kinaseinhibitors have been disclosed in WO2011/143426 and WO2010/071837(Vertex).

According to one aspect of the invention there is provided a compound ofthe Formula (I)

wherein:R¹ is methyl or ethyl; andR² is (C2-3)alkyl substituted by hydroxyl;or a pharmaceutically-acceptable salt thereof.

In another aspect of the invention, there is provided a compound ofFormula (I) as defined above.

It will be understood that the term “(C2-3)alkyl substituted by hydroxy”includes both straight chain and branched alkyl groups, for examplethose illustrated as groups (i) to (xi) below:

It is to be understood that, insofar as certain of the compounds ofFormula (I) defined above may exist in optically active or racemic formsby virtue of one or more asymmetric carbon atoms, the invention includesin its definition any such optically active or racemic form whichpossesses PI3K-α and -δ inhibitory activity. The synthesis of opticallyactive forms may be carried out by standard techniques of organicchemistry well known in the art, for example by synthesis from opticallyactive starting materials or by resolution of a racemic form. Similarly,the above-mentioned activity may be evaluated using the standardlaboratory techniques.

A particular enantiomer of a compound described herein may be moreactive that other enantiomers of the same compound.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically-acceptable saltthereof, which is a single enantiomer being in an enantiomeric excess (%ee) of ≧95, ≧98% or ≧99%. Conveniently, the single enantiomer is presentin an enantiomeric excess (% ee) of ≧99%.

According to a further aspect of the invention there is provided apharmaceutical composition, which comprises a compound of the Formula(I), which is a single enantiomer being in an enantiomeric excess (% ee)of ≧95, ≧98% or ≧99% or a pharmaceutically-acceptable salt thereof, inassociation with a pharmaceutically-acceptable diluent or carrier.Conveniently, the single enantiomer is present in an enantiomeric excess(% ee) of ≧99%.

Some compounds of Formula (I) may be crystalline and may have more thanone crystalline form. It is to be understood that the present inventionencompasses any crystalline or amorphous form, or mixtures thereof,which form possesses properties useful in the inhibition of PI3K-α and-δ activity, it being well known in the art how to determine efficacy ofa crystalline or amorphous form for the inhibition of PI3K-α and/or -δactivity by the standard tests described hereinafter.

It is generally known that crystalline materials may be analysed usingconventional techniques such as X-Ray Powder Diffraction (hereinafterXRPD) analysis, Differential Scanning Calorimetry (hereinafter DSC),Thermal Gravimetric Analysis (hereinafter TGA), Diffuse ReflectanceInfrared Fourier Transform (DRIFT) spectroscopy, Near Infrared (NIR)spectroscopy, solution and/or solid state nuclear magnetic resonancespectroscopy. The water content of such crystalline materials may bedetermined by Karl Fischer analysis.

As an example, the compound of Example 1 exhibits crystallinity and onecrystalline form has been identified.

Accordingly, a further aspect of the invention is Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=5.1°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=18.0°.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with at least two specificpeaks at about 2-theta=5.1 and 18.0°.

According to a further aspect of the present invention there is provideda crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with specific peaks atabout 2-theta=5.1, 18.0, 10.2, 11.7, 19.4, 18.5, 14.8, 26.7, 26.6,17.8°.

According to the present invention there is provided crystalline form,Form A which has an X-ray powder diffraction pattern substantially thesame as the X-ray powder diffraction pattern shown in FIG. 1.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=5.1° plus or minus 0.2° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=18.0° plus or minus 0.2° 2-theta.

According to a further aspect of the present invention, there isprovided a crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with at least two specificpeaks at about 2-theta=5.1 and 18.0° plus or minus 0.2° 2-theta.

According to a further aspect of the present invention there is provideda crystalline form, Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one,which has an X-ray powder diffraction pattern with specific peaks atabout 2-theta=5.1, 18.0, 10.2, 11.7, 19.4, 18.5, 14.8, 26.7, 26.6, 17.8°plus or minus 0.2° 2-theta.

Example 3 is also crystalline and three forms (A, B and C) are describedherein.

According to the present invention there is provided a crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=4.8°.

According to the present invention there is provided a crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=10.0°.

According to the present invention there is provided a crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least two specificpeaks at about 2-theta=4.8° and 10.0°.

According to the present invention there is provided a crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with specific peaks atabout 2-theta=4.8, 10.0, 14.6, 5.2, 19.9, 10.4, 25.4, 23.6, 24.4, 16.2°.

According to the present invention there is provided crystalline form,Form A of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern substantially the same asthe X-ray powder diffraction pattern shown in FIG. 3.

According to the present invention there is provided crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=4.8° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=10.0° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least two specificpeaks at 2-theta=4.8° and 10.0° wherein said values may be plus or minus0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form A, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with specific peaks at2-theta=4.8, 10.0, 14.6, 5.2, 19.9, 10.4, 25.4, 23.6, 24.4, 16.2°wherein said values may be plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=5.8°.

According to the present invention there is provided a crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=10.9°.

According to the present invention there is provided a crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least two specificpeaks at about 2-theta=5.8° and 10.9°.

According to the present invention there is provided a crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with specific peaks atabout 2-theta=5.8, 10.9, 11.5, 25.9, 17.3, 24.0, 19.1, 12.9, 24.7,27.2°.

According to the present invention there is provided crystalline form,Form B of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern substantially the same asthe X-ray powder diffraction pattern shown in FIG. 5.

According to the present invention there is provided crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=5.8° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=10.9° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least two specificpeaks at 2-theta=5.8° and 10.9° wherein said values may be plus or minus0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form B, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with specific peaks at2-theta=5.8, 10.9, 11.5, 25.9, 17.3, 24.0, 19.1, 12.9, 24.7, 27.2°wherein said values may be plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=6.9°.

According to the present invention there is provided a crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at about 2-theta=12.3°.

According to the present invention there is provided a crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least two specificpeaks at about 2-theta=6.9° and 12.3°.

According to the present invention there is provided a crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with specific peaks atabout 2-theta=6.9, 12.3, 10.5, 21.0, 24.6, 13.6, 16.4, 19.6, 20.2,22.5°.

According to the present invention there is provided crystalline form,Form C of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern substantially the same asthe X-ray powder diffraction pattern shown in FIG. 7.

According to the present invention there is provided crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=6.9° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least one specificpeak at 2-theta=12.3° plus or minus 0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with at least two specificpeaks at 2-theta=6.9° and 12.3° wherein said values may be plus or minus0.2° 2-theta.

According to the present invention there is provided a crystalline form,Form C, of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-onewhich has an X-ray powder diffraction pattern with specific peaks at2-theta=6.9, 12.3, 10.5, 21.0, 24.6, 13.6, 16.4, 19.6, 20.2, 22.5°wherein said values may be plus or minus 0.2° 2-theta.

When it is stated that the present invention relates to a crystallineform of a compound of the invention, such as Example 1 or Example 3, thedegree of crystallinity is conveniently greater than about 60%, moreconveniently greater than about 80%, preferably greater than about 90%and more preferably greater than about 95%. Most preferably the degreeof crystallinity is greater than about 98%.

When it is stated that the present invention relates to a crystallineform of a compound of the invention, such as Example 1 or Example 3, thecrystalline form is preferably substantially free of other crystallineforms or amorphous form of the same compound. In this context,“substantially free” conveniently means greater than about 60%, moreconveniently greater than about 80%, preferably greater than about 90%,more preferably greater than about 95%, still more preferable greaterthan about 98% and even more preferably greater than about 99% puresingle crystalline form. For example, Example 3 may be in the form ofForm A and substantially free of forms B and C; alternatively, Example 3may be in the form of Form B and substantially free of forms A and C;alternatively Example 3 may be in the form of Form C and substantiallyfree of forms A and B. Similarly, Example 3 may be in the form of Form Band substantially free of alternative crystalline or amorphous forms.

It will be understood that 2-theta values of the X-ray powderdiffraction patterns may vary slightly from one machine to another orfrom one sample to another, and so the values quoted are not to beconstrued as absolute.

It is known that an X-ray powder diffraction pattern may be obtainedwhich has one or more measurement errors depending on measurementconditions (such as equipment or machine used). In particular, it isgenerally known that intensities in an X-ray powder diffraction patternmay fluctuate depending on measurement conditions. Therefore it shouldbe understood that the crystalline Forms of the present inventiondescribed above, unless otherwise stated, are not limited to thecrystals that provide X-ray powder diffraction patterns identical to theX-ray powder diffraction pattern shown in FIGS. 1, 3, 5 and any crystalsproviding X-ray powder diffraction patterns substantially the same asthose shown in these Figures fall within the scope of the presentinvention. A person skilled in the art of X-ray powder diffraction isable to judge the substantial identity of X-ray powder diffractionpatterns.

Persons skilled in the art of X-ray powder diffraction will also realisethat the relative intensity of peaks can be affected by, for example,grains above 30 microns in size and non-unitary aspect ratios, which mayaffect analysis of samples. The skilled person will also realise thatthe position of reflections can be affected by the precise height atwhich the sample sits in the diffractometer and the zero calibration ofthe diffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values (see Jenkins, R & Snyder, R. L. ‘Introductionto X-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C. W.(1948), Chemical Crystallography, Clarendon Press, London; Klug, H. P. &Alexander, L. E. (1974), X-Ray Diffraction Procedures).

Generally, a measurement error of a diffraction angle in an X-ray powderdiffractogram is approximately plus or minus 0.2° 2-theta, and suchdegree of a measurement error should be taken into account whenconsidering the X-ray powder diffraction data. Furthermore, it should beunderstood that intensities might fluctuate depending on experimentalconditions and sample preparation (preferred orientation).

Particular compounds of the invention are each of the Examples, each ofwhich provides a further independent aspect of the invention. Furtherparticular compounds of the invention are pharmaceutically-acceptablesalt(s) of each of the Examples, each of which provides a furtherindependent aspect of the invention.

According to a further aspect of the invention there is provided acompound of the Formula (I), which is obtainable by following any of theExamples as disclosed herein.

A further feature is any of the scopes defined herein with the provisothat specific Examples, such as Example 1, 3, 4 etc. are individuallydisclaimed.

It will be appreciated by those skilled in the art that certaincompounds of Formula (I) contain asymmetrically substituted carbonatoms, and accordingly may exist in, and be isolated in,optically-active and racemic forms. Some compounds of Formula (I) mayexhibit polymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic or stereoisomericform, or mixtures thereof, which form possesses properties useful in theinhibition of PI3K-α and -δ activity, it being well known in the art howto prepare optically-active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, by enzymaticresolution, by biotransformation, or by chromatographic separation usinga chiral stationary phase) and how to determine efficacy for theinhibition of PI3K-α and -δ activity by the standard tests describedhereinafter.

It is to be understood that certain compounds of Formula (I) definedabove may exhibit the phenomenon of tautomerism. It is to be understoodthat the present invention includes in its definition any suchtautomeric form, or a mixture thereof, which possesses PI3K inhibitoryactivity and is not to be limited merely to any one tautomeric formutilised within the formulae drawings or named in the Examples. Ingeneral, just one of any such tautomeric forms is named in the Examplesthat follow hereinafter or is presented in any relevant formulaedrawings that follow hereinafter.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes will be understood toinclude those atoms having the same atomic number but different massnumbers. For example, isotopes of hydrogen include tritium anddeuterium. Isotopes of carbon include ¹³C and ¹⁴C.

A suitable pharmaceutically-acceptable salt of a compound of the Formula(I) is, for example, an acid-addition salt of a compound of the Formula(I), for example an acid-addition salt with a strong inorganic ororganic acid such as hydrochloric, hydrobromic, sulphuric ortrifluoroacetic acid. A further suitable pharmaceutically-acceptablesalt of a compound of the Formula (I) is, for example, a salt formedwithin the human or animal body after administration of a compound ofthe Formula (I).

It is further to be understood that a suitablepharmaceutically-acceptable solvate of a compound of the Formula (I)also forms an aspect of the present invention. A suitablepharmaceutically-acceptable solvate is, for example, a hydrate such as ahemi-hydrate, a mono-hydrate, a di-hydrate or a tri-hydrate or analternative quantity thereof.

It is further to be understood that a suitablepharmaceutically-acceptable pro-drug of a compound of the Formula (I)also forms an aspect of the present invention. Accordingly, thecompounds of the invention may be administered in the form of apro-drug, which is a compound that is broken down in the human or animalbody to release a compound of the invention. A pro-drug may be used toalter the physical properties and/or the pharmacokinetic properties of acompound of the invention. A pro-drug can be formed when the compound ofthe invention contains a suitable group or substituent to which aproperty-modifying group can be attached. Examples of pro-drugs includein-vivo cleavable ester derivatives that may be formed at a hydroxygroup in a compound of the Formula (I), and in-vivo cleavable amidederivatives that may be formed at an amino group in a compound ofFormula (I).

Accordingly, the present invention includes those compounds of theFormula (I) as defined hereinbefore when made available by organicsynthesis and when made available within the human or animal body by wayof cleavage of a pro-drug thereof. Accordingly, the present inventionincludes those compounds of the Formula (I) that are produced by organicsynthetic means and also such compounds that are produced in the humanor animal body by way of metabolism of a precursor compound, that is acompound of the Formula (I) may be a synthetically-produced compound ora metabolically-produced compound.

A suitable pharmaceutically-acceptable pro-drug of a compound of theFormula (I) is one that is based on reasonable medical judgement asbeing suitable for administration to the human or animal body withoutundesirable pharmacological activities and without undue toxicity.

Various forms of pro-drug have been described, for example in thefollowing documents:—

-   a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder,    et al. (Academic Press, 1985);-   b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);-   c) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and    Application of Pro-drugs”, by H. Bundgaard p. 113-191 (1991);-   d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);-   e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285    (1988);-   f) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984);-   g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”,    A.C.S. Symposium Series, Volume 14; and-   h) E. Roche (editor), “Bioreversible Carriers in Drug Design”,    Pergamon Press, 1987.

A suitable pharmaceutically-acceptable pro-drug of a compound of theFormula (I) that possesses a hydroxy group is, for example, an in vivocleavable ester or ether thereof. An in vivo cleavable ester or ether ofa compound of the Formula (I) containing a hydroxy group is, forexample, a pharmaceutically-acceptable ester or ether which is cleavedin the human or animal body to produce the parent hydroxy compound.Suitable pharmaceutically-acceptable ester forming groups for a hydroxygroup include inorganic esters such as phosphate esters (includingphosphoramidic cyclic esters). Further suitablepharmaceutically-acceptable ester forming groups for a hydroxy groupinclude (1-10C)alkanoyl groups such as acetyl, benzoyl, phenylacetyl andsubstituted benzoyl and phenylacetyl groups, (1-10C)alkoxycarbonylgroups such as ethoxycarbonyl, N,N-[di-(1-4C)alkyl]carbamoyl,2-dialkylaminoacetyl and 2-carboxyacetyl groups. Examples of ringsubstituents on the phenylacetyl and benzoyl groups include aminomethyl,N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl,piperazin-1-ylmethyl and 4-(1-4C)alkylpiperazin-1-ylmethyl. Suitablepharmaceutically-acceptable ether forming groups for a hydroxy groupinclude α-acyloxyalkyl groups such as acetoxymethyl andpivaloyloxymethyl groups.

A suitable pharmaceutically-acceptable pro-drug of a compound of theFormula (I) that possesses an amino group is, for example, an in vivocleavable amide derivative thereof. Suitable pharmaceutically-acceptableamides from an amino group include, for example an amide formed with(1-10C)alkanoyl groups such as an acetyl, benzoyl, phenylacetyl andsubstituted benzoyl and phenylacetyl groups. Examples of ringsubstituents on the phenylacetyl and benzoyl groups include aminomethyl,N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl,piperazin-1-ylmethyl and 4-(1-4C)alkylpiperazin-1-ylmethyl.

The in vivo effects of a compound of the Formula (I) may be exerted inpart by one or more metabolites that are formed within the human oranimal body after administration of a compound of the Formula (I). Asstated hereinbefore, the in vivo effects of a compound of the Formula(I) may also be exerted by way of metabolism of a precursor compound (apro-drug).

Compounds of Formula (I) contain a piperidine sub-unit substituted by—C(O)R², wherein R² is (C2-3)alkyl substituted by hydroxyl. Onepotential route of metabolism of these compounds is by oxidation of thehydroxyl substituent on this group. These oxidised compounds generallyretain some PI3K-α and -δ inhibitory activity.

Therefore, according to a further aspect of the invention there isprovided a compound of the formula (A):

wherein:R^(1A) is methyl or ethyl; andR^(2A) is (C1-2)alkyl substituted by carboxy;or a pharmaceutically-acceptable salt thereof.

Examples of compounds of Formula (A) include Example 8, which is anidentified metabolite of Example 1.

and Example 9 which is an identified metabolite of Example 3:

Further potential metabolites of Example 3 are two alternative oxidationproducts, shown below and further described in Examples 10 and 11:

Suitable pharmaceutically-acceptable salts of compounds of formula (A)include for example an alkali or alkaline earth metal salt such as acalcium or magnesium salt, or an ammonium salt, or a salt with anorganic base such as methylamine, dimethylamine, trimethylamine,piperidine, morpholine or tris-(2-hydroxyethyl)amine.

For the avoidance of doubt it is to be understood that where in thisspecification a group is qualified by ‘hereinbefore defined’ or ‘definedhereinbefore’ the said group encompasses the first occurring andbroadest definition as well as each and all of the particulardefinitions for that group.

Particular novel compounds of the invention include, for example,compounds of the Formula (I), or pharmaceutically-acceptable saltsthereof, wherein, unless otherwise stated, each of R¹ and R², has any ofthe meanings defined hereinbefore or in the following statements:

R¹ is methyl.

R¹ is ethyl.

R² is any of groups (i) to (xi) as hereinbefore defined.

R² is groups (i) to (vi) as hereinbefore defined.

R² is group (i).

A particular group of compounds of the invention are compounds ofFormula (I) above wherein:—

R¹ is methyl or ethyl,R² is group (i):

or a pharmaceutically-acceptable salt thereof.

Particular compounds of the invention are, for example, the compounds ofthe Formula (I) that are disclosed within the Examples that are set outhereinafter.

For example, a particular compound of the invention is a compound of theFormula (I) selected from any one of the following:—

-   1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one    (Example 1 and 2);-   1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one    (Example 3);-   (3R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one    (Example 4);-   (3S)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one    (Example 5);-   (2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-2-methyl-propan-1-one    (Example 6);-   1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-2-hydroxy-2-methyl-propan-1-one    (Example 7).

Another aspect of the present invention provides a process for preparinga compound of the Formula (I), or a pharmaceutically-acceptable saltthereof. A suitable process is illustrated by the followingrepresentative process variants in which, unless otherwise stated, R¹,R² have any of the meanings defined hereinbefore. Necessary startingmaterials may be obtained by standard procedures of organic chemistry.The preparation of such starting materials is described in conjunctionwith the following representative process variants and within theaccompanying Examples. Alternatively, necessary starting materials areobtainable by analogous procedures to those illustrated which are withinthe ordinary skill of an organic chemist.

Suitable process variants include, for example, the following:

(a) The reaction, conveniently in the presence of a suitable activatingreagent, of a compound of the Formula II

wherein R¹ has any of the meanings defined hereinbefore, with thecarboxylic acid R²—COOH except that any functional group is protected ifnecessary, in the presence of a suitable base, whereafter any protectinggroup that is present is removed.

Suitable coupling agents for this reaction include for example,O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, TBTU(2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouroniumtetrafluoroborate) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride ion the presence of 2-hydroxy-pyridine N-oxide.

The reaction is conveniently carried out in the presence of a suitablebase. A suitable base is, for example, an organic amine base such as,for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine,triethylamine, N-methylmorpholine, diazabicyclo[5.4.0]undec-7-ene,diisopropylethyl amine, or, for example, an alkali or alkaline earthmetal carbonate or hydroxide, for example sodium carbonate, potassiumcarbonate, calcium carbonate, sodium hydroxide or potassium hydroxide;preferably N-ethyl-N,N-diisopropylamine.

The reaction is conveniently carried out in the presence of a suitableinert solvent such as for example, acetonitrile, N,N-dimethylformamide,N-methylpyrrolidone, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane,benzene, toluene, xylene, methanol, ethanol, halogenated solvents suchas dichloromethane, chloroform or carbon tetrachloride and at atemperature in the range, for example −50° C. to 100° C., preferably inthe range 0° C. to 30° C.

Alternatively, the carboxylic acid R²—COOH may be transformed into anactivated species, which can then be reacted with a compound of theFormula II under conditions well known in the art.

A suitable protecting group for the hydroxyl group is thetetrahydropyran protecting group, as described in Example 2 and 3.

Suitable conditions for removing this group include mild acidicconditions in the presence of an alcohol as the solvent at temperaturebetween 20 to 70° C., such as methanol or ethanol. A typical mild acidused is pyridine p-toluenesulfonate.

A compound of Formula II can be obtained from reaction of compound ofFormula III:

where P is a protecting group, such as tert-butoxycarbonyl, with ancompound of Formula R¹-L when L is a suitable leaving group such as forexample, a halogeno group such as a bromo, iodo group (convenientlyiodo), in the presence of a suitable base, whereafter any protectinggroup that is present is removed.

A suitable base is, for example, an organic amine base, such as1,8-diazabicyclo[5.4.0]undec-7-ene.

The reaction is conveniently carried out in the presence of a suitableinert solvent such as for example 2-methyltetrahydrofuran,tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene,xylene, and at a temperature in the range, for example −50° C. to 60°C., preferably in the range −10° C. to 0° C.

Suitable conditions for deprotection of the tert-butoxycarbonyl includeacidic conditions such as trifluoroacetic acid in an inert solvent suchas dichloromethane at approximately room temperature (20-25° C.).

Compound III can be obtained from a coupling reaction in the presence ofa suitable activating reagent, of compound of Formula IV

with a compound of Formula V

preferably in the presence of a suitable base, followed by a cyclisationreaction in the presence of a mild acid.

The coupling reaction can be carried out in the presence of a suitablecoupling agent such as, for example,O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate or TBTU(2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouroniumtetrafluoroborate).

The coupling reaction is conveniently carried out in the presence of asuitable base. A suitable base is, for example, an organic amine basesuch as, for example, pyridine, 2,6-lutidine, collidine,4-dimethylaminopyridine, triethylamine, N-methylmorpholine,diazabicyclo[5.4.0]undec-7-ene, diisopropylethyl amine, or, for example,an alkali or alkaline earth metal carbonate or hydroxide, for examplesodium carbonate, potassium carbonate, calcium carbonate, sodiumhydroxide or potassium hydroxide; preferablyN-ethyl-N,N-diisopropylamine.

The coupling reaction is conveniently carried out in the presence of asuitable inert solvent such as for example, N,N-dimethylacetamide,N,N-dimethylformamide, N-methylpyrrolidone, tetrahydrofuran,1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene, xylene, methanol,ethanol, halogenated solvents such as dichloromethane, chloroform orcarbon tetrachloride and at a temperature in the range, for example −50°C. to 100° C., preferably in the range 0° C. to 30° C.

The cyclisation conditions are carried out in the presence of a mildacid, typically acetic acid. The reaction is conveniently carried out inthe presence of a suitable inert solvent such as for example,N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone,tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene,xylene at a temperature in the range, for example 50° C. to 150° C.,preferably in the range 80° C. to 100° C.

Compound IV can be obtained from a reaction of compound of Formula VIwith hydrazine.

This reaction is conveniently carried out in the presence of a suitableinert solvent such as for example tetrahydrofuran, 1,4-dioxane,1,2-dimethoxyethane, benzene, toluene, xylene or an alcohol such asethanol or isopropanol at a temperature in the range, for example 20° C.to 70° C., preferably around 50° C.

Compound VI can be obtained from a metal-catalysed reaction of compoundof Formula VII with a source of cyanide such zinc (II) dicyanide.

A suitable catalyst for the reaction includes, for example, a metalliccatalyst such as palladium(0), for exampletetrakis(triphenylphosphine)palladium(0); or a catalyst formed in-situfrom a palladium (II) salt, for example palladium(II) acetate,palladium(II) chloride, palladium(II) bromide,bis(triphenylphosphine)palladium(II) chloride,[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), ortris(dibenzilideneacetone)dipalladium, and a phosphine ligand, forexample, dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine. Thereaction is conveniently carried out in a suitable solvent such as,N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran,1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene or xylene and at atemperature in the range, for example 20° C. to 150° C., preferably inthe range 60° C. to 120° C. The reaction is also conveniently carriedout in the presence of additional metal, such as zinc.

Suitable reactions of this type are described in ‘Metal-CatalyzedCross-Coupling Reactions’, Second Edition, Edited by Armin Meijere,Francois Diederich, Wiley-VCH, 2004).

Syntheses of Compound VII have been described in Examples 1 and 2.

Alternatively, a compound of Formula II can be obtained bymetal-catalysed reaction of compound VIII, where R is a small alkyl andcompound IX, where P is a protecting group, such as tert-butoxycarbonyl,

A suitable catalyst for the reaction includes, for example, a metalliccatalyst such as palladium(0), for exampletetrakis(triphenylphosphine)palladium(0); or a catalyst formed in-situfrom a palladium (II) salt, for example palladium(II) acetate,palladium(II) chloride, palladium(II) bromide,bis(triphenylphosphine)palladium(II) chloride,[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), ortris(dibenzilideneacetone)dipalladium, and a phosphine ligand, forexample, dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine.

The reaction is conveniently carried out in a suitable solvent such as,N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran,1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene or xylene or analcohol such as 4-methyl-2-pentanol at a temperature in the range, forexample 50° C. to 180° C., preferably in the range 120° C. to 150° C.

The reaction is also conveniently carried out in the presence ofadditional salt such as lithium chloride.

Suitable reactions of this type are described in ‘Metal-CatalyzedCross-Coupling Reactions’, Second Edition, Edited by Armin Meijere,Frangois Diederich, Wiley-VCH, 2004).

A compound of Formula VIII can be obtained from metal-catalysed reactionof compound VII with a suitable hexa-alkyl distannane. A suitablecatalyst for the reaction includes, for example, a metallic catalystsuch as palladium(0), for exampletetrakis(triphenylphosphine)palladium(0); or a catalyst formed in-situfrom a palladium (II) salt, for example palladium(II) acetate,palladium(II) chloride, palladium(II) bromide,bis(triphenylphosphine)palladium(II) chloride,[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), ortris(dibenzilideneacetone)dipalladium, and a phosphine ligand, forexample, dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine.

The reaction is conveniently carried out in a suitable solvent such as,N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran,1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene, xylene or an alcoholsuch as 4-methyl-2-pentanol at a temperature in the range, for example50° C. to 100° C., preferably in the range 70° C. to 80° C.

Compound of Formula IX can be obtained from commercially availablematerial in a few steps, as illustrated in Example 1 (with R¹=Me andP=tert-butoxycarbonyl).

It is to be understood that other permutations of the process steps inthe process variants described above are also possible.

It is to be understood that any compound of Formula (I) obtained by anyof the processes described hereinbefore can be converted into anothercompound of the Formula (I) if required.

When a pharmaceutically-acceptable salt of a compound of the Formula (I)is required, for example an acid-addition salt, it may be obtained by,for example, reaction of said compound with a suitable acid.

When a pharmaceutically-acceptable pro-drug of a compound of the Formula(I) is required, it may be obtained using a conventional procedure. Forexample, an in vivo cleavable ester of compound of the Formula (I) maybe obtained by, for example, reaction of a compound of the Formula (I)containing a hydroxy group with a pharmaceutically-acceptable carboxylicacid. Further information on pro-drugs has ben provided hereinbefore.

It will also be appreciated that, in some of the reactions mentionedhereinbefore, it may be necessary or desirable to protect any sensitivegroups in the compounds. The instances where protection is necessary ordesirable, and suitable methods for protection, are known to thoseskilled in the art. Conventional protecting groups may be used inaccordance with standard practice (for illustration see T. W. Green,Protective Groups in Organic Synthesis, John Wiley and Sons, 1991).Thus, if reactants include groups such as amino, carboxy or hydroxy, itmay be desirable to protect the group in some of the reactions mentionedherein.

A suitable protecting group for an amino or alkylamino group is, forexample, an acyl group, for example an alkanoyl group such as acetyl, analkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl ort-butoxycarbonyl group, an arylmethoxycarbonyl group, for examplebenzyloxycarbonyl, or an aroyl group, for example benzoyl. Thedeprotection conditions for the above protecting groups necessarily varywith the choice of protecting group. Thus, for example, an acyl groupsuch as an alkanoyl or alkoxycarbonyl group or an aroyl group may beremoved for example, by hydrolysis with a suitable base such as analkali metal hydroxide, for example lithium or sodium hydroxide.Alternatively an acyl group such as a t-butoxycarbonyl group may beremoved, for example, by treatment with a suitable acid as hydrochloric,sulphuric or phosphoric acid or trifluoroacetic acid and anarylmethoxycarbonyl group such as a benzyloxycarbonyl group may beremoved, for example, by hydrogenation over a catalyst such aspalladium-on-carbon, or by treatment with a Lewis acid for example borontris(trifluoroacetate). A suitable alternative protecting group for aprimary amino group is, for example, a phthaloyl group which may beremoved by treatment with an alkylamine, for exampledimethylaminopropylamine, or with hydrazine.

A suitable protecting group for a hydroxy group is, for example, an acylgroup, for example an alkanoyl group such as acetyl, an aroyl group, forexample benzoyl, or an arylmethyl group, for example benzyl. Thedeprotection conditions for the above protecting groups will necessarilyvary with the choice of protecting group. Thus, for example, an acylgroup such as an alkanoyl or an aroyl group may be removed, for example,by hydrolysis with a suitable base such as an alkali metal hydroxide,for example lithium or sodium hydroxide. Alternatively an arylmethylgroup such as a benzyl group may be removed, for example, byhydrogenation over a catalyst such as palladium-on-carbon.

A suitable protecting group for a carboxy group is, for example, anesterifying group, for example a methyl or an ethyl group which may beremoved, for example, by hydrolysis with a base such as sodiumhydroxide, or for example a t-butyl group which may be removed, forexample, by treatment with an acid, for example an organic acid such astrifluoroacetic acid, or for example a benzyl group which may beremoved, for example, by hydrogenation over a catalyst such aspalladium-on-carbon.

The protecting groups may be removed at any convenient stage in thesynthesis using conventional techniques well known in the chemical art.

Certain of the intermediates (for example, compounds of the Formulae II,III, IV, VI, VII, VIII) defined herein are novel and these are providedas a further feature of the invention.

Biological Assays—

The following assays were used to measure the effects of the compoundsof the present invention as a) inhibitors of PI3-kinase enzymes inbiochemical assays, b) as inhibitors of other kinases in biochemicalassays, c) as inhibitors in vitro of phospho AKT (Thr308) in BT474cells, d) as inhibitors in vitro of phospho AKT (Ser473) in MDA-MB-468cells, e) as inhibitors in vitro of phospho AKT (Ser473) in JEKO cells,f) as inhibitors in vitro of phospho Chk1 (Ser345) in HT29 cells, g) asinhibitors of cell proliferation across a panel of tumour cell lines, h& i) as inhibitors in vivo of phospho AKT (Ser473) or inhibitors in vivoof tumour growth respectively, in SCID mice transplanted with the humanbreast adenocarcinoma cell line, MCF7.

Abbreviations used in Assay Protocols:

-   PIP2: PI(4,5)P2, phosphatidyl inositol 4,5-bisphosphate-   s.c.: sub-cutaneously-   ATP: Adenosine triphosphate-   DMSO: Dimethyl sulphoxide-   TRIS: Tris(Hydroxymethyl)aminomethane-   CHAPS: 3-[(3-Cholamidopropyl)dimethylammonio]-1 l-propanesulfonate-   DTT: Dithiothreitol-   FBS: Foetal bovine serum-   DMEM: Dulbecco's Modified Eagle Medium-   EDTA: Ethylenediaminetetraacetic acid-   EGTA: Ethylene glycol tetraacetic acid-   BSA: Bovine Serum albumin-   PBS: Phosphate buffered saline-   HRP: Horseradish peroxidase-   RPMI: Roswell Park Memorial Institute 1640 medium-   4NQO: 4-Nitroquinoline N-oxide-   EMEM: Eagle's Minimal Essential medium-   CO₂: Carbon dioxide-   PBST: Phosphate buffered saline/Tween-   Ab: Antibody-   MTS reagent:    [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,    inner salt; MTS] and an electron coupling reagent (phenazine    methosulfate) PMS.

(a) In Vitro Enzyme Inhibition Assay

The inhibition of PI3K-β, PI3K-α, PI3K-γ and PI3K-δ was evaluated in aKinase Glo based enzyme activity assay using human recombinant enzymes.The assay platform indirectly measured the depletion of ATP afterincubation with enzyme, PIP2 substrate, ATP and compound.

After completion of the enzyme reaction the remaining ATP was used in asecondary enzymatic reaction, where Luciferase converted beetleluciferin into oxyluciferin under the emission of light. A directrelationship existed between the luminescence measured and the ATPremaining in a completed kinase reaction. Therefore, the luminescencewas inversely related to the kinase activity. Typically, twelvedifferent compound concentrations were tested and raw data from theinhibition of PI3K-β, PI3K-α, PI3K-γ or PI3K-δ were plotted versusinhibitor concentration.

Method Details:

Compounds in 100% DMSO were added to assay plates by acousticdispensing. PI3K enzyme was added in a Tris buffer (50 mM Tris pH7.4,0.05% CHAPS, 2.1 mM DTT, and 10 mM magnesium chloride) and allowed topreincubate with compound for 20 minutes prior to addition of substratesolution containing PIP2 and ATP. The enzyme reaction was stopped after80 minutes by the addition of Kinase Glo detection solution containingLucferin and Luciferase (from Kinase Glo(R) Plus Luminecent Kinase Assaykit (Promega #V3772). Plates were left for 30 minutes at roomtemperature then read on a Pherastar Instrument with a standardLuminescence filter block. The final concentration of DMSO, ATP and PIP2in the assay were, 1%, 8 μM, and 80 μM respectively.

Data Analysis

IC₅₀ values were calculated using a log curve fitting to a non-linearregression fit. The IC₅₀ value was the concentration of test compoundthat inhibited 50% of enzyme activity.

(b) Evaluation of Kinase Selectivity, Beyond PI3Kinase Class 1 Enzymes

Large panels of kinase assays are offered by a range of commercialvendors such as Millipore, Invitrogen and ProQinase. Such panels allowfor an assessment of the overall kinase selectivity of a given compound.The precise methods/technologies will vary depending on the vendor.

Selectivity data for some of the compounds described herein wasgenerated using enzyme assays performed at the MRC—Division of SignalTransduction Therapy (DSTT), MRC Protein Phosphorylation Unit, Dundee,UK. Protein kinase assays were carried using a Radiochemical format.Assays were performed in multidrop 384 well plates at room temperaturein a total assay volume of 25.5 μl. Compounds were pre-incubated in thepresence of the enzyme and peptide/protein substrate for 5 minutesbefore initiation of the reaction by addition of 10 μl of ATP (finalconcentration selected for each kinase at 5, 20 or 50 μM). Assays wererun at room temperature before termination by the addition of 5 μlorthophosphoric acid. The assay plate contents were then harvested ontoWhatman-P81-Unifilter Plates by a Packard Harvester (wash buffer was 50mM orthophosphoric acid) and dried in air. The dry Unifilter plates werethen sealed on the addition of MicroScint O and were counted in PackardTopcount NXT scintillation counters. This protocol captures the genericformat suitable for the majority of kinases in the panel, butmodifications to the protocols were required for a small number ofkinases, as will be familiar to those skilled in the art.

Lipid kinase assays for ˜18 lipid kinases were also performed at DSTT.All lipid kinase assays were carried out in 384 well plates at roomtemperature in a total assay volume of 40 μl. The assay was performedaccording to the protocols provided with the ADP-GLO assay (Promega,#V9101). This protocol captures the generic format suitable for themajority of kinases in the panel, but modifications to the protocolswere required for a small number of kinases, as will be familiar tothose skilled in the art.

Kinase selectivity was also evaluated using the KINOMEscan™ screeningplatform, available via DiscoverX. This employs an active site-directedcompetition binding assay to quantitatively measure interactions betweentest compounds and more than 450 human kinases and disease relevantmutant variants. KINOMEscan™ assays do not require ATP and therebyreport true thermodynamic interaction affinities, as opposed to IC50values, which can depend on the ATP concentration. The methodology isbased on compounds that bind the kinase active site and directly(sterically) or indirectly (allosterically) preventing kinase binding tothe immobilized ligand, thereby reducing the amount of kinase capturedonto a solid support. Conversely, test molecules that do not bind thekinase have no effect on the amount of kinase captured on a solidsupport. Screening “hits” are identified by measuring the amount ofkinase captured in test versus control samples by using a quantitativeqPCR method that detects the associated DNA label. In a similar manner,dissociation constants (Kds) for test compound-kinase interactions arecalculated by measuring the amount of kinase captured on the solidsupport as a function of the test compound concentration.

(c) Protocol for Assay Measuring Phosphorylated AKT (Tyr308) in BT474Cells

This assay was used to measure PI3K-α inhibition in cells. BT474 cells(human breast ductal carcinoma, ATCC HTB-20) were seeded into black 384well plates (Costar, #3712) at a density of 5600 cells/well in DMEMcontaining 10% FBS and 1% glutamine and allowed to adhere overnight.

The following morning compounds in 100% DMSO were added to assay platesby acoustic dispensing. After a 2 hour incubation at 37° C. and 5% CO₂,the medium was aspirated and the cells were lysed with a buffercontaining 25 mM Tris, 3 mM EDTA, 3 mM EGTA, 50 mM sodium fluoride, 2 mMSodium orthovanadate, 0.27M sucrose, 10 mM 3-glycerophosphate, 5 mMsodium pyrophosphate, 0.5% Triton X-100 and complete protease inhibitorcocktail tablets (Roche #04 693 116 001, used 1 tab per 50 ml lysisbuffer).

After 20 minutes, the cell lysates were transferred into ELISA plates(Greiner #781077) which had been pre-coated with an anti total-AKTantibody in PBS buffer and non-specific binding was blocked with 1% BSAin PBS containing 0.05% Tween 20. Plates were incubated over night at 4°C. The next day the plates were washed with PBS buffer containing 0.05%Tween 20 and further incubated with a mouse monoclonal anti-phospho AKTT308 for 2 hours. Plates were washed again as above before addition of ahorse anti-mouse-HRP conjugated secondary antibody. Following a 2 hourincubation at room temperature, plates were washed and QuantaBlusubstrate working solution (Thermo Scientific #15169, prepared accordingto provider instruction) was added to each well. The developedfluorescent product was stopped after 60 minutes by addition of Stopsolution to the wells. Plates were read using a Tecan Safire platereader using 325 nm excitation and 420 nm emission wavelengthsrespectively. Except where specified, reagents contained in the PathScan Phospho AKT (Thr308) sandwich ELISA kit from Cell Signalling(#7144) were used in this ELISA assay.

(d) Protocol for Detection of Phospho AKT (Ser473) in MDA-MB-468 Cellsas a Measure for PI3Kinase-Beta Inhibition.

This assay was used to measure PI3K-β inhibition in cells, and was used,in conjunction with assay (c) above, to determine alpha vs betaselectivity in cells. MDA-MB-468 cells (human breast adenocarcinoma#ATCC HTB 132) were seeded at 1500 cells/well in 40 μl of DMEMcontaining 10% FBS and 1% glutamine into Greiner 384 well blackflat-bottomed plates. Cell plates were incubated for 18 hours in a 37°C. incubator before dosing with compounds in 100% DMSO using acousticdispensing.

Compounds were dosed in a 12 point concentration range into a randomisedplate map. Control wells were generated either by dosing of 100% DMSO(max signal) or addition of a reference compound (a PI3K-β inhibitor)that completely eliminated the pAKT signal (min control). Plates wereincubated at 37° C. for 2 hours, cells were then fixed by the additionof 10 μl of a 3.7% formaldehyde solution. After 30 minutes the plateswere washed with PBS using a Tecan PW384 plate washer. Wells wereblocked and cells permeabilised with the addition of 40 μl of PBScontaining 0.5% Tween20 and 1% Marvel™ (dried milk powder) and incubatedfor 60 minutes at room temperature. The plates were washed with PBScontaining 0.5% (v/v) Tween20 and 20 μl rabbit anti-phospho AKT Ser473(Cell Signalling Technologies, #3787) in same PBS-Tween+1% Marvel™ wasadded and incubated overnight at 4° C.

Plates were washed 3 times with PBS+0.05% Tween 20 using a Tecan PW384.20 μl of secondary antibody Alexa Fluor 488 anti-Rabbit (MolecularProbes, #A11008) diluted in PBS+0.05% Tween20 containing 1% Marvel™ wasadded to each well and incubated for 1 hour at room temperature. Plateswere washed three times as before then 20 μl PBS added to each well andplates sealed with a black plate sealer.

The plates were read on an Acumen plate reader as soon as possible,measuring green fluorescence after excitation with 488 nm laser. Usingthis system IC₅₀ values were generated and quality of plates wasdetermined by control wells. Reference compounds were run each time tomonitor assay performance.

(e) Protocol for Detection of Phospho AKT (Ser473) in Jeko Cells Thisassay was used to measure PI3K-δ inhibition in cells. Compounds at ×10final concentration in 10 μl of 1% (v/v) DMSO were added to the wells ofa Greiner V-bottomed 96 well plate (Sigma #M9686). Compounds were dosedin a 10-point concentration range from top dose of 1 μM or 10 μM, 8compounds were dosed on one plate. There were 8 maximum signal controlwells per plate dosed with anti-IgM (AffiniPure F(ab′)2 Fragment GoatAnti-Human IgM (Stratech, #109-006-129) and vehicle, and 8 minimumsignal control wells dosed with anti-IgM and a reference PI3K-δinhibitor. Final vehicle concentration was 0.1% DMSO. A full doseresponse curve for a PI3K-δ selective compound was included in each run.Jeko B cells (human mantle cell lymphoma, ATCC #CRL-3006) were seededinto the Greiner 96 well V-bottomed plates containing compounds. Cellswere seeded at 100,000 cell/well in 70 μl of RPMI containing 1%glutamine.

Cell plates were incubated with compound for 1 hour in a 37° C.incubator. After this compound pre-incubation time, the above describedanti-IgM was added to the plates at ×5 final concentration in 20 μl ofassay buffer (RPMI containing 1% glutamine). Final anti-IgMconcentration was 0.06 μg/ml or an equivalent EC90 dose. Plates wereincubated at 37° C. for 10 min, then plates were immediately placed onice and centrifuged at 12000 rpm for 4 min. On ice, supernatants werecarefully removed with a manual pipette and 40 μl lysis buffer added.Plates were incubated on ice for 5 min and stored at −80° C. untilassayed in the phosphor (Ser473)/total Akt whole cell lysate kitaccording to manufacturer's instructions (Mesoscale Diagnostics,#K11100D-3).

(f) Protocol for detection of phospho Chk1 (Ser 345) in HT29 cells

ATR (Ataxia Telangiectasia+Rad3-related kinase) is a PI3-kinase-relatedkinase which phosphorylates multiple substrates on serine or threonineresidues in response to DNA damage or replication blocks. Chk1, adownstream protein kinase of ATR, plays a key role in DNA damagecheckpoint control. Activation of Chk1 involves phosphorylation ofSer317 and Ser345 (the latter regarded as the preferential target forphosphorylation/activation by ATR).

This was a cell based assay to measure inhibition of ATR kinase, bymeasuring a decrease in phosphorylation of Chk1 (Ser 345) in HT29 cells,following treatment with compound and the UV mimetic 4NQO (Sigma#N8141). HT29 cells (ECACC #85061109) were seeded into 384 well assayplates (Costar #3712) at a density of 6000 cells/well in 40 μl EMEMmedium containing 1% L glutamine and 10% FBS and allowed to adhereovernight. The following morning compounds in 100% DMSO were added toassay plates by acoustic dispensing. After 1 hour incubation at 37° C.and 5% CO₂, 40 nl of 3 mM 4NQO in 100% DMSO was added to all wells byacoustic dispensing, except minimum control wells which were leftuntreated with 4NQO to generate a null response control. Plates werereturned to the incubator for a further 1 hour. Then cells were fixed byadding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20mins at room temperature. Then 20 μl of 0.1% Triton X100 in PBS wasadded and incubated for 10 minutes at room temperature, to permeabalisecells. Then the plates were washed once with 50 μl/well PBS, using aBiotek EL405 plate washer.

Phospho-Chk1 Ser 345 antibody (Cell Signalling Technology #2348) wasdiluted 150 fold in PBS containing 0.05% polysorbate/Tween and 15 μl wasadded to each well and incubated over night at room temperature. Thenext morning plates were washed three times with 50 μl/well PBS, using aBiotek EL405 plate washer, and then 20 μl of secondary Ab solution,containing 500 fold diluted Alexa Fluor 488 Goat anti-rabbit IgG(Molecular Probes #A-11008) and 0.002 mg/ml Hoeschst dye (MolecularProbes #H-3570), in PBST, was added. After 2 hours incubation at roomtemperature, the plates were washed three times with 50 μl/well PBS,using a Biotek EL405 plate washer, and plates were then sealed withblack plate seals until read. Plates were read using an ArrayScan VTIinstrument, using an XF53 filter with 10× objective. A two laser set upwas used to analyse nuclear staining with Hoeschst (405 nM) andsecondary antibody staining of pChk1 (488 nM).

(g) Cell Proliferation Assays in Tumour Cell Lines (Used to Demonstratea Personalised Medicine Hypothesis)

The sensitivity of a panel of human cancer cell lines to the effects ofcompounds was determined in a standard proliferation assay. Cell lineswere sourced through the AstraZeneca Cell Bank. The majority of celllines are also available through Cell Bank Repositories known to thoseworking in the art, for example ATCC, ECACC, DMSZ, RIKEN, KCLB, JCRB(HSRRB), LBNL, CLS and ICLC.

Cells were plated in 96 well plates at densities of 1000-6000 cells perwell in RPMI media containing 10% FBS. After incubation at 37° C. for 16hours, various concentrations of compound were added to the assayplates. After incubation for an additional 72 h, the viable cells weredetermined by the addition of MTS reagent (Promega #3582) to each wellfor 2 h. MTS is a tetrazolium salt that is bioreduced by metabolicallyactive cells in the presence of an electron coupling reagent toformazan. The formazan product was then quantitated by absorbance at 490nm, as an indicator of the relative number of live cells. In order todetermine the GI50 (concentration at which growth of cells was inhibitedby 50%) the relative number of cells present at the time of drugaddition was determined by comparison with the MTS readout before thedrug was added, and this value was subtracted from the 72 hr value ofuntreated cells as a measure of cell growth during the assay.

Analysis of this data, described below under ‘PersonalisedHealthcare/Personalised Medicine Examples’ illustrates how this data maybe analysed to reveal that PI3Kα inhibitors display selective growthinhibition of cell lines with PIK3CA gene mutation. This illustrates aPersonalised Healthcare (PHC) or Personalised Medicine opportunity wherea response prediction biomarker readout could be used to identifypatients with tumours containing mutation in the PIK3CA gene and whowould be more likely to respond to the compounds described herein.

Other potential markers of response to the compounds described hereininclude, but are not limited to, increased copy number, amplification ortranslocation of the PIK3CA gene, and other genetic, genomic orproteomic changes which provide a measure of PI33-Kinase pathwayactivation or dependency; for example but not limited to, activation ofone or more receptor tyrosine kinases, or mutation or translocation inthe PIK3R genes which encode the regulatory subunits (p85) ofPI3-Kinase, or phosphorylation of downstream signalling markers such aspAKT, pS6, or FOXO status. In addition, analysis of further genes and/orthe signalling of their protein products, for example Kras, may helpimprove the predictivity of a Personalised Medicine approach.

(h) Protocol for Detection of Phospho AKT (Ser473) from MCF-7 TumoursGrown in Male SCID Mice

This was a pharmacodynamic assay providing a measure of PI3K-αinhibition in an animal model. Male SCID mice (AZ UK, also availablefrom Charles River, UK) were transplanted sub-cutaneously (s.c.) withhuman breast adenocarcinoma cell line MCF7 (ICRF London, also availablefrom ATCC #HTB-22)) to determine the inhibition of phosphorylation ofAKT with PI3-kinase inhibitors. Mice were implanted with a 0.5 mg 21-dayoestrogen pellet (Innovative Research of America, #E121) 24 hours priorto cell implantation. 5×10⁶ cells in 50% matrigel (BD Bioscience) wereinjected s.c. on the left flank of the animals. Animals were randomisedinto groups of 8 control and 4 treatment when tumours reached a volumeof 400 mm³ and dosing commenced the next day. Tumours were taken atselected time points, when blood samples were also taken for PKmeasurements.

Tumours excised from mouse were placed into a Fast Prep tube (2 mlridged tubes containing lysing matrix A, MP Biomedicals #6910-500) andimmediately snap frozen. 1 ml of lysis buffer (25 mM Tris, 3 mM EDTA, 3mM EGTA, 50 mM sodium fluoride, 2 mM orthovanadate, 0.27M sucrose, 10 mMbeta-glycerophoshate, 5 mM pyrophosphate, 0.5% Triton x-100) plusphosphatase inhibitors (Sigma #P2850 and Sigma #P5726, diluted 1:100)and protease inhibitors (Sigma #P8340, diluted 1:200) were added to eachtube. The tumours were homogenised for 1 minute on a FastPrep-TM machine(MP Biomedicals #116004500) and then left on ice for 5 minutes, followedby two further homogenisations steps, each followed by a 5 minutesincubation on ice. Samples were spun for 10 minutes at 13,000 rpm in achilled centrifuge. Cleared lysates were then taken into fresh tubes and10 μl used for a protein determination assay

The detection of total and phosphorylated AKT (ser473) was carried outusing a MSD multi-spot assay kit (Meso Scale Discovery #K15100D-3). Eachwell of the plate contained 4 spots; two of these were coated with mousemonoclonal antibodies provided with the kit; one was coated with acapture antibody for total AKT and one was coated with an antibody forphosphorylated AKT (ser473). The plates were blocked overnight in thecold room on a shaker with 150 μl of blocking solution per well, whichwas made using 20 ml of 1× solution of wash solution plus 600 mg BlockerA supplied with the kit. Plates were washed three time with 0.3 ml perwell of wash solution. An aliquot of the lysate was taken from eachtumour and diluted to a concentration of 2 mg/ml with lysis buffer, 25μl of the diluted lysate was then added to each well giving a totalamount of 50 μg per well. The plates were placed on a shaker at roomtemperature for one hour before plates were washed three times. Adetection antibody solution was prepared using a mix of blocking andwash solution plus a 1 in 50 dilution of the 50×SULFO-TAG-TM anti-totalAKT antibody. The plates were placed on a shaker at room temperature forone hour before plates were washed three times. 150 μl of read buffersupplied with the kit was diluted 1:4 with deionised water and added toeach well and then the plate was read on MSD plate analyser. The readbuffer gives the correct chemical environment forelectrochemiluminescence, so that when the plate reader applies avoltage to the plate the electrodes on the base of the plate cause thelabel bound to the detection antibody to emit light. The intensity oflight emitted is a quantitative measure of the AKT, either total orphosphorylated, that is present. To calculate the ratio ofphosphorylated to total AKT a calculation was applied as suggested byMeso Scale: two times phosphorylated signal divided by total plusphosphorylated signal then multiplied by 100 to give % phosphoprotein.The values were converted into Log 10, and then these values were usedto calculate the Geomean for each group plus standard error. A student Ttest was then applied using 2 tailed formula and unequal variance tocheck for significance. Studies showed that a control group of 8 animalswith 4 per treatment group were sufficient to power the study.

(i) Protocol for Detection of Tumour Growth Inhibition in Human BreastAdenocarcinoma Cell Line MCF7 Transplanted into SCID Mice.

This method provides for assessment of anti-tumour efficacy ofPI3-kinase inhibitors in vivo, in a PI3K-α dependent model. As for thePD studies, indicated above, male SCID mice were transplanted s.c. withhuman breast adenocarcinoma cell line, MCF7. Mice were implanted with a0.5 mg 21-day oestrogen pellet 24 hours prior to cell implantation.5×10⁶ cells in 50% matrigel were injected s.c. on the left flank of theanimals. Animals were randomised into groups of 10-15 when tumoursreached a volume of ˜200-300 mm³ and treatment commenced. Animals weredosed for 2-4 weeks by peroral, intravenous or intra-peritoneal routeswith compound in a vehicle suitable for dosing via the required routeand consistent with welfare requirements (suspension for oral dosing inpH range 4-7, solution for ip/iv dosing in pH range 5.5-7.0) and atdefined doses. Tumours were usually measured twice weekly by caliper andvolume of tumours calculated using elliptical formula(pi/6×width×width×length).

Although the pharmacological properties of the compounds of the Formula(I) vary with structural change as expected, in general activitypossessed by compounds of the Formula (I) may be demonstrated at thefollowing concentrations or doses in one or more of the above tests (a)and (c):—

-   -   Test (a):—IC₅₀ versus PI3K-α in the range, for example, 1 nM-100        nM;    -   Test (c):—IC₅₀ versus cellular phospho AKT (Tyr308) in BT474        cells, in the range, for example, 10 nM-1 μM;

Conveniently, particular compounds of the invention possess activity atthe following concentrations or doses in one or more of the above tests(a) and (c):—

-   -   Test (a):—IC₅₀ versus PI3K-α in the range, for example, 1 nM-100        nM;    -   Test (c):—IC₅₀ versus cellular phospho AKT (Tyr308) in BT474        cells, in the range, for example, 10 nM-1 μM;

Conveniently, particular compounds of the invention possess activity atthe following concentrations or doses in one or more of the above tests(a), (c), (h) and (i):—

-   -   Test (a):—IC₅₀ versus PI3K-α in the range, for example, 1 nM-100        nM;    -   Test (c):—IC₅₀ versus cellular phospho AKT (Tyr308) in BT474        cells, in the range, for example, 10 nM-1 μM;    -   Test (h):—>50% inhibition of in vivo phospho AKT (ser473) in the        range, for example, 1-200 mg/kg/day;    -   Test (i):—xenograft activity in the range, for example, 1-200        mg/kg/day.        The following data were generated for the Examples:

TABLE A PI3K-α PI3K-δ PI3K-β PI3K-α cell ATR cell Example inhibitionIC₅₀ inhibitionIC₅₀ inhibitionIC₅₀ IC₅₀ IC₅₀ number (μM)* (μM)* (μM)*(μM) ** (μM)# 1 0.023 <0.014 2.24 0.36 >30 3 0.007 <0.010 0.57 0.09 >304 0.025 <0.012 2.91 0.31 >30 5 0.030 0.012 3.31 0.27 >30 6 0.032 <0.0123.42 0.53 >30 7 0.037 0.014 6.26 0.42 >30 8 0.024 0.012 1.52 0.59 >30 9<0.010 <0.010 0.640 0.33 — 10 — — — 0.085 — 11 — — — 0.11 — *Testprotocol a: these are mean values calculated from a number of replicatesof the test. ** Test protocol c: these are mean values calculated from anumber of replicates of the test. #Test protocol f: one test replicateonly carried out.

Combination Studies Materials and Methods

MCF7 is an estrogen receptor positive breast tumour cell line carrying amutation in the PIKC3CA gene (E545K). Male SCID mice (AZ UK) weretransplanted subcutaneously (s.c.) with human breast adenocarcinoma cellline MCF7 (ICRF London) to determine anti-tumour activity of PI3 kinaseinhibitors. Mice were implanted with a 0.5 mg 21 day oestrogen pellet(Innovative Research of America) 24 hours prior to cell implantation.5×10⁶ cells in 50% matrigel (BD Bioscience) were injected s.c. on theleft flank of the animals.

BT474 is an estrogen receptor positive breast tumour cell lines withelevated Her2 expression and carries a mutation in the PIK3CA gene(K111N). Female Swiss athymic nude mice (swiss nu/nu—AZUK) weretransplanted subcutaneously with human epithelial breast ductalcarcinoma cell line BT474c (derived in AZ from BT474—ATCC HTB-20)tumours passaged in mice. Mice were implanted with 0.36 mg 60 dayoestrogen pellet (Innovative Research of America) 24 hours prior to cellimplantation. 5×10⁶ cells in 50% matrigel (BD Bioscience) were injecteds.c. on the left flank of the animals.

HCC70 is breast tumour cell line which is deficient in PTEN geneexpression. Female Swiss athymic nude mice (swiss nu/nu—AZUK) weretransplanted subcutaneously with the breast ductal epithelial tumourcell line HCC70 (ATCC—CRL2315) cells. 1×10⁶ cells in 50% matrigel (BDBioscience) were injected s.c. on the left flank of the animals.

Animals were randomised into groups of 10-15 when tumours reached avolume of ˜200-300 mm³ and treatment commenced. Animals were dosed for3-4 weeks by peroral route, with compound in a suitable vehicle atdefined doses and schedules. Tumours were measured two-three timesweekly by caliper and volume of tumours calculated using ellipticalformula (pi/6×width×width×length).

When dosed alone, AZD5363 was formulated in 10% DMSO, 25% Kleptosesolution. (Kleptose is sourced from Roquette—Pharma (Trademarked)Hydroxypropyl betacyclodextrin—suitable for in vivo use andformulations).

When co-dosed with Example 3, AZD5363 was formulated in HPMC/Tween (0.5%Methocel (hydroxypropyl methocellulose)/0.1% Polysorbate 80). Thesuspension was ball milled overnight.

Example 3 was formulated in HPMC/Tween (0.5% Methocel (hydroxypropylmethocellulose)/0.1% Polysorbate 80).

AZD8186 was formulated in HPMC/Tween (0.5% Methocel (hydroxypropylmethocellulose)/0.1% Polysorbate 80).

When co-dosed with Example 3, AZD8186 was formulated in HPMC/Tween (0.5%Methocel (hydroxypropyl methocellulose)/0.1% Polysorbate 80). Thesuspension was ball milled overnight.

Olaparib was formulated in 10% DMSO/30% Kleptose solution.

Tumour Growth Inhibition by Example 3 in Combination with AKT Inhibitor(AZD5363)—Sequential Administration

Studies were performed in the BT474 xenograft model. Example 3 andAZD5363 were dosed twice daily (BID) 6-8 hours apart on a 2 days on/5days off weekly cycle, in sequence such that AZD5363 was dosed on days 1and 2 of the weekly cycle and Example 3 was dosed on days 3 and 4 of theweekly cycle. Example 3 was dosed at 50 mg/kg BID and AZD5363 was dosedat 170 mg/kg BID, in HPMC/Tween and DMSO/Kleptose respectively.

The tumour growth curve (shown in FIG. 9) indicates that intermittentdosing of either Example 3 or AZD5363 partially inhibited tumour growthrelative to vehicle only control (HPMC/Tween). The combination ofExample 3 plus AZD5363 induced tumour regression.

Tumour Growth Inhibition by Example 3 in Combination with AKT Inhibitor(AZD5363)—Co-Administration

Studies were performed in the BT474 xenograft model. Example 3 andAZD5363 were dosed twice daily (BID) 6-8 hours apart and concomitantlyon a 2 days on/5 days off weekly cycle. Example 3 was dosed at 25 mg/kgBID and AZD5363 was dosed at 100 mg/kg BID, both in HPMC/Tween.

The tumour growth curve (shown in FIG. 10) indicates that intermittentdosing of either Example 3 or AZD5363 partially inhibited tumour growthrelative to vehicle only control (HPMC/Tween). The combination ofExample 3 plus AZD5363 induced tumour regression during the dosingperiod, although followed by tumour re-growth during the dosing freeperiod.

Tumour Growth Inhibition by Example 3 in Combination with PARP Inhibitor(Olaparib)

Studies were performed in the BT474 xenograft model. Example 3 andOlaparib were dosed on every day throughout the study, Example 3 twicedaily (BID) 6-8 hours apart at 25 mg/kg each dose and Olaparib oncedaily (QD) at 100 mg/kg 1 hour post first daily dose of Example 3. Bothagents were dosed in HPMC/Tween.

The tumour growth curve (FIG. 11) indicates that olaparib alone had nosignificant effect on tumour growth, example 3 alone partially inhibitedgrowth, but the combination of Example 3 plus olaparib induced tumourregression.

Tumour Growth Inhibition by Example 3 in Combination with PARP Inhibitor(Olaparib)

Studies were performed in the MCF7 xenograft model. Example 3 andOlaparib were dosed on every day throughout the study, Example 3 twicedaily 6-8 hours apart at 25 mg/kg each dose and Olaparib once daily (QD)at 100 mg/kg 1 hour post first daily dose of Example 3. Both agents weredosed in HPMC/Tween.

The tumour growth curve (FIG. 12) indicates that olaparib alone hadminimal effect on tumour growth, Example 3 alone caused some tumourregression, but the combination of Example 3 plus olaparib inducedstronger tumour regression.

Tumour Growth Inhibition by Example 3 in Combination with PI3Kbeta/DeltaInhibitor (AZD8186)

Studies were performed in the HCC70 xenograft model. Example 3 andAZD8186 were dosed on every day, twice daily (BID), throughout thestudy, Example 3 at 25 mg/kg each dose and AZD8186 at 50 mg/kg eachdose. Both agents were dosed in HPMC/Tween. The tumour growth curve(FIG. 13) indicates that AZD8186 partially inhibited tumour growth,Example 3 alone inhibited growth more strongly, but the combination ofExample 3 plus AZD8186 induced tumour regression.

According to a further aspect of the invention there is provided apharmaceutical composition, which comprises a compound of the Formula(I), or a pharmaceutically-acceptable salt thereof, as definedhereinbefore in association with a pharmaceutically-acceptable diluentor carrier.

Suitable pharmaceutically acceptable excipients for a tablet formulationinclude, for example, inert diluents, granulating and disintegratingagents, binding agents, lubricating agents, preservative agents andanti-oxidants. Tablet formulations may be uncoated or coated either tomodify their disintegration and the subsequent absorption of the activeingredient within the gastrointestinal tract, or to improve theirstability and/or appearance, in either case, using conventional coatingagents and procedures well known in the art.

Compositions for oral use may alternatively be in the form of hardgelatin capsules in which the active ingredient is mixed with an inertsolid diluent, or as soft gelatin capsules in which the activeingredient is mixed with water or an oil.

Aqueous suspensions generally contain the active ingredient in finelypowdered form together with one or more suspending agents, dispersing orwetting agents. The aqueous suspensions may also contain one or morepreservatives, anti-oxidants, colouring agents, flavouring agents,and/or sweetening agents.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil or in a mineral oil. The oily suspensions may alsocontain a thickening agent. Sweetening agents such as those set outabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Additional excipients such as sweetening,flavouring and colouring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil or amineral oil or a mixture of any of these. The emulsions may also containsweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents, and mayalso contain a demulcent, preservative, flavouring and/or colouringagent.

The pharmaceutical compositions may also be in the form of a sterileinjectable aqueous or oily suspension, which may be formulated accordingto known procedures using one or more of the appropriate dispersing orwetting agents and suspending agents, which have been mentioned above. Asterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent.

Compositions for administration by inhalation may be in the form of aconventional pressurised aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may be used and the aerosoldevice is conveniently arranged to dispense a metered quantity of activeingredient.

For further information on formulation the reader is referred to Chapter25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, oral administration to humans willgenerally require, for example, from 1 mg to 2 g of active agent (moresuitably from 100 mg to 2 g, for example from 250 mg to 1.8 g, such asfrom 500 mg to 1.8 g, particularly from 500 mg to 1.5 g, convenientlyfrom 500 mg to 1 g) to be administered compounded with an appropriateand convenient amount of excipients which may vary from about 3 to about98 percent by weight of the total composition. It will be understoodthat, if a large dosage is required, multiple dosage forms may berequired, for example two or more tablets or capsules, with the dose ofactive ingredient divided conveniently between them. Conveniently, asingle solid dosage form may contain between 1 and 300 mg of activeingredient.

The size of the dose for therapeutic or prophylactic purposes of acompound of the Formula (I) will naturally vary according to the natureand severity of the disease state, the age and sex of the animal orpatient and the route of administration, according to well knownprinciples of medicine.

In using a compound of the Formula (I) for therapeutic or prophylacticpurposes it will generally be administered so that a daily dose in therange, for example, 1 mg/kg to 100 mg/kg body weight is received, givenif required in divided doses. In general, lower doses will beadministered when a parenteral route is employed. Thus, for example, forintravenous administration, a dose in the range, for example, 1 mg/kg to25 mg/kg body weight will generally be used. Similarly, foradministration by inhalation, a dose in the range, for example, 1 mg/kgto 25 mg/kg body weight will be used. Oral administration is howeverpreferred, particularly in tablet form. Typically, unit dosage formswill contain about 10 mg to 0.5 g of a compound of this invention.

The compounds of the invention may be administered daily or more thanonce daily. The compounds of the invention may also be administered in asuitable dosing schedule, for example the compounds of the invention maybe administered one or more times per day (for example once, twice orthree times a day) for a certain number of days, followed by a period ofdays where no dose is given. This dosage cycle (consisting of dosingdays and no-dosing days) may then be repeated. Conveniently a dosagecycle is a period of 5-14 days, such as 5, 7, 10 or 14 days, moreconveniently 7 days. In one aspect, the compounds of formula (I) aredosed for 1 day or 2 or 3 consecutive days, followed by 3, 4, 5 or 6days with no dose in a dosage cycle.

In one aspect the compound of formula (I) is dosed for 1 day followed byno dose for 2, 3 or 4 days.

In another aspect the compound of formula (I) is dosed for 2 daysfollowed by no dose for 4, 5 or 6 days.

In a further aspect the compound of formula (I) is dosed for 3 daysfollowed by no dose for 3, 4 or 5 days.

In another aspect the compound of formula (I) is dosed for 4 daysfollowed by no dose for 2, 3 or 4 days.

In another aspect the compound of formula (I) is dosed for 5 daysfollowed by no dose for 1, 2 or 3 days.

In another aspect, the compound of formula (I) is dosed every other day.

The above dosing schedules are conveniently applied when the compoundsof the invention are used as monotherapy. Further examples of potentialdosing schedules for administration of the compounds of the invention ascombination therapy are described hereinafter.

As stated above, it is known that PI3K-α and -δ enzymes contribute totumourigenesis by one or more of the effects of mediating proliferationof cancer and other cells, mediating angiogenic events and mediating themotility, migration and invasiveness of cancer cells. We have found thatthe compounds of the present invention possess potent anti-tumouractivity which it is believed is obtained by way of inhibition of PI3K-αand -δ enzymes that are involved in the signal transduction steps whichlead to the proliferation and survival of tumour cells and theinvasiveness and migratory ability of metastasising tumour cells.

Accordingly, the compounds of the present invention are of value asanti-tumour agents, in particular as selective inhibitors of theproliferation, survival, motility, dissemination and invasiveness ofmammalian cancer cells leading to inhibition of tumour growth andsurvival and to inhibition of metastatic tumour growth. Particularly,the compounds of the present invention are of value asanti-proliferative and anti-invasive agents in the containment and/ortreatment of solid tumour disease. Particularly, the compounds of thepresent invention are expected to be useful in the prevention ortreatment of those tumours which are sensitive to inhibition of PI3K-αand/or -δ enzymes and that are involved in the signal transduction stepswhich lead to the proliferation and survival of tumour cells and themigratory ability and invasiveness of metastasising tumour cells.Further, the compounds of the present invention are expected to beuseful in the prevention or treatment of those tumours which aremediated alone or in part by inhibition of PI3K-α and/or -δ enzymes,i.e. the compounds may be used to produce a PI3K-α and/or -δ enzymeinhibitory effect in a warm blooded animal in need of such treatment.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use as a medicament in awarm-blooded animal such as man.

According to a further aspect of the invention, there is provided acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use in the production of ananti-proliferative effect in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a compound of the Formula (I), or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for use in a warm-blooded animalsuch as man as an anti-invasive agent in the containment and/ortreatment of solid tumour disease.

According to a further aspect of the invention, there is provided theuse of a compound of the Formula (I), or a pharmaceutically acceptablesalt thereof, as defined hereinbefore for the production of ananti-proliferative effect in a warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the manufacture of amedicament for use in the production of an anti-proliferative effect ina warm-blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided the use of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the manufacture of amedicament for use in a warm-blooded animal such as man as ananti-invasive agent in the containment and/or treatment of solid tumourdisease.

According to a further feature of this aspect of the invention there isprovided a method for producing an anti-proliferative effect in a warmblooded animal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound of theFormula (I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further feature of this aspect of the invention there isprovided a method for producing an anti-invasive effect by thecontainment and/or treatment of solid tumour disease in a warm bloodedanimal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound of theFormula (I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention, there is provided acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use in the prevention or treatmentof cancer in a warm blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the prevention or treatment of cancer in a warm blooded animalsuch as man.

According to a further feature of this aspect of the invention there isprovided a method for the prevention or treatment of cancer in a warmblooded animal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound of theFormula (I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further aspect of the invention, there is provided acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use in the prevention or treatmentof solid tumour disease in a warm blooded animal such as man.

According to a further aspect of the invention there is provided the useof a compound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore in the manufacture of a medicament foruse in the prevention or treatment of solid tumour disease in a warmblooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method for the prevention or treatment of solid tumourdisease in a warm blooded animal, such as man, in need of such treatmentwhich comprises administering to said animal an effective amount of acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use in the prevention or treatmentof those tumours which are sensitive to inhibition of PI3K-α and/or -δenzymes that are involved in the signal transduction steps which lead tothe proliferation, survival, invasiveness and migratory ability oftumour cells.

According to a further feature of this aspect of the invention there isprovided the use of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the manufacture of amedicament for use in the prevention or treatment of those tumours whichare sensitive to inhibition of PI3K-α and/or -δ enzymes that areinvolved in the signal transduction steps which lead to theproliferation, survival, invasiveness and migratory ability of tumourcells.

According to a further feature of this aspect of the invention there isprovided a method for the prevention or treatment of those tumours whichare sensitive to inhibition of PI3K-α and/or -δ enzymes that areinvolved in the signal transduction steps which lead to theproliferation, survival, invasiveness and migratory ability of tumourcells which comprises administering to said animal an effective amountof a compound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore.

According to a further aspect of the invention there is provided acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined hereinbefore for use in providing a PI3K-α and -δenzyme inhibitory effect.

According to a further feature of this aspect of the invention there isprovided the use of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the manufacture of amedicament for use in providing a PI3K-α and -δ enzyme inhibitoryeffect.

According to a further aspect of the invention there is also provided amethod for providing a PI3K-α and -δ enzyme inhibitory effect whichcomprises administering an effective amount of a compound of the Formula(I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

As stated hereinbefore, certain compounds of the present inventionpossess substantially better potency against PI3K-α and -δ enzymes thanagainst other PI3-kinase enzymes or other kinases. Such compoundspossess sufficient potency against PI3K-α and -δ enzymes that they maybe used in an amount sufficient to inhibit PI3K-α and -δ enzymes whilstdemonstrating little activity against the PI3K-β enzyme and against mostother kinase enzymes. Such compounds are likely to be useful for theselective inhibition of PI3K-α and -δ enzymes and are likely to beuseful for the effective treatment of, for example PI3K-α and/or -δenzyme driven tumours.

According to this aspect of the invention there is provided a compoundof the Formula (I), or a pharmaceutically acceptable salt thereof, asdefined hereinbefore for use in providing a selective PI3K-α and -δenzyme inhibitory effect.

According to a further feature of this aspect of the invention there isprovided the use of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore in the manufacture of amedicament for use in providing a selective PI3K-α and -δ enzymeinhibitory effect.

According to a further aspect of the invention there is also provided amethod for providing a selective PI3K-α and -δ enzyme inhibitory effectwhich comprises administering an effective amount of a compound of theFormula (I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

By “a selective PI3K-α and -δ enzyme inhibitory effect” is meant thatthe compound of the Formula (I) are more potent against PI3K-α and -δenzymes than against other class 1 PI3-kinases, and generally displaygood selectivity relative to other members of the wider PI3-kinasefamily and across the broader classes of kinase enzymes comprisingtyrosine and ser/thr kinases.

According to a further feature of the invention there is provided acompound of the Formula (I), or a pharmaceutically acceptable saltthereof, as defined herein before for use in the treatment of cancer ofthe Breast, Stomach (Gastric) and Oesophagus cancers, Non Small CellLung Cancer (NSCLC) including squamous cell carcinomas (SCC) andadenocarcinoma, SCC of the Head and Neck (H&N), Gynaecological cancers(including Endometrial, Ovarian and Cervical) and of Haematologicalcancers such as multiple myeloma, lymphomas and leukemias (includingChronic Lymphoctyic Leukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL)and Mantle Cell Lymphoma (MCL).

According to a further feature of this aspect of the invention there isprovided a compound of the Formula (I), or a pharmaceutically acceptablesalt thereof, as defined herein before for use in the treatment ofcancer of the Bladder, Brain/CNS, Colorectum, Lung (all other forms),Gallbladder and Bile duct, and Skin.

According to a further feature of this aspect of the invention there isprovided a compound of the Formula (I), or a pharmaceutically acceptablesalt thereof, as defined herein before for use in the treatment ofcancer of the Prostate, Bone, Kidney, Liver, Melanoma, Gastrointestinaltissue, Pancreas, Testes, Thyroid, Penile, Vulva, and other tumour typeswith a PI3-kinase dependency through mutation, amplification or otheraberrations.

According to a further feature of this aspect of the invention there isprovided a method for treating cancer of Breast, Stomach (Gastric) andOesophagus cancers, NSCLC including SCC and adenocarcinoma, SCC of H&N,Gynaecological cancers (including Endometrial, Ovarian and Cervical) andof Haematological cancers such as multiple myeloma, lymphomas andleukemias (including CLL, ALL and MCL) in a warm blooded animal such asman that is in need of such treatment which comprises administering aneffective amount of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined hereinbefore.

According to a further feature of this aspect of the invention there isprovided a method for treating cancer of Bladder, Brain/CNS, Colorectum,Lung (all other forms), Gallbladder and Bile duct, and Skin in a warmblooded animal such as man that is in need of such treatment whichcomprises administering an effective amount of a compound of the Formula(I), or a pharmaceutically acceptable salt thereof, as definedhereinbefore.

According to a further feature of this aspect of the invention there isprovided a method for treating cancer of Prostate, Bone, Kidney, Liver,Melanoma, Gastrointestinal tissue, Pancreas, Testes, Thyroid, Penile,Vulva, and other tumour types with a PI3-kinase dependency throughmutation, amplification or other aberrations, in a warm blooded animalsuch as man that is in need of such treatment which comprisesadministering an effective amount of a compound of the Formula (I), or apharmaceutically acceptable salt thereof, as defined hereinbefore.

According to a further feature of the invention there is provided theuse of a compound of the Formula (I), or a pharmaceutically acceptablesalt thereof, as defined herein before in the manufacture of amedicament for use in the treatment of cancer of the Breast, Stomach(Gastric) and Oesophagus cancers, NSCLC including SCC andadenocarcinoma, SCC of H&N, Gynaecological cancers (includingEndometrial, Ovarian and Cervical) and of Haematological cancers such asmultiple myeloma, lymphomas and leukemias (including CLL, ALL and MCL).

According to a further feature of this aspect of the invention there isprovided the use of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined herein before in the manufacture ofa medicament for use in the treatment of cancer of the Bladder,Brain/CNS, Colorectum, Lung (all other forms), Gallbladder and Bileduct, and Skin.

According to a further feature of this aspect of the invention there isprovided the use of a compound of the Formula (I), or a pharmaceuticallyacceptable salt thereof, as defined herein before in the manufacture ofa medicament for use in the treatment of cancer of the of Prostate,Bone, Kidney, Liver, Melanoma, Gastrointestinal tissue, Pancreas,Testes, Thyroid, Penile, Vulva, and other tumour types with a PI3-kinasedependency through mutation, amplification or other aberrations.

In one feature of the invention, the cancer to be treated is breastcancer. In a further aspect of this feature, the breast cancer isEstrogen Receptor+ve. In one embodiment of this aspect, the compound ofFormula (I), or a pharmaceutically acceptable salt thereof, is dosed incombination with an anti-hormonal agent as defined herein. In anotherembodiment of this aspect, Example 3 is dosed in combination with ananti-hormonal agent as defined herein. In a further embodiment of thisaspect, Example 3 is dosed in combination with olaparib, or apharmaceutically-acceptable salt thereof, and optionally further incombination with an anti-hormonal agent as defined herein. In a furtherembodiment of this aspect, Example 3 is dosed in combination withAZD5363, or a pharmaceutically-acceptable salt thereof, and optionallyfurther in combination with an anti-hormonal agent as defined herein.

In one aspect where the treatment of cancer is indicated, it is to beunderstood that this may refer to the prevention of metastases and thetreatment of metastases, i.e. cancer spread. Therefore the compounds ofthe present invention might be used to treat a patient who has nometastases to stop them occurring, or to lengthen the time period beforethey occur, and to a patient who already has metastases to treat themetastases themselves. Furthermore the treatment of cancer may refer totreatment of an established primary tumour or tumours and developingprimary tumour or tumours. Therefore, in one aspect the treatment ofcancer relates to the prevention of metastases. In another aspect of theinvention the treatment of cancer relates to the treatment ofmetastases. In another aspect of the invention the treatment of cancerrelates to treatment of an established primary tumour or tumours ordeveloping primary tumour or tumours.

As stated hereinbefore, the in vivo effects of a compound of the Formula(I) may be exerted in part by one or more metabolites (such as compoundsof formula A as defined hereinbefore) that are formed within the humanor animal body after administration of a compound of the Formula (I).

Particular compounds of the invention possess better potency againstPI3-kinase-α and -δ than against other class I PI3-kinase isoforms suchas -β and -γ. In one aspect the compounds of the invention are selectivefor PI3K-α and -δ compared to PI3K-β or -γ.

The present invention therefore also contemplates a method forinhibiting PI3-kinase-α in a patient, comprising administering to apatient an amount of the compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, effective in inhibiting the phosphoinositide3-kinase-α in the patient.

The present invention therefore also contemplates a method forinhibiting PI3-kinase-α and -δ in a patient, comprising administering toa patient an amount of the compound of Formula (I), or apharmaceutically acceptable salt thereof, effective in inhibiting thePI3-kinase-α and -δ in the patient.

The compound of Formula (I), or a pharmaceutically acceptable saltthereof, being an inhibitor of PI3-kinase, also has potentialtherapeutic uses in a variety of other disease states. For example,PI3-kinase plays an important role in promoting smooth muscleproliferation in the vascular tree, i.e. vascular smooth muscle cells(Thyberg, European Journal of Cell Biology, 1998, 76(1), 33-42), and inthe lungs (airway smooth muscle cells), Krymskaya, V. P., BioDrugs,2007, 21(2), 85-95. Excessive proliferation of vascular smooth musclecells plays an important role in the formation of atheroscleroticplaques and in the development of neointimal hyperplasia followinginvasive vascular procedures (Scwartz et al., Progress in CardiovascularDisease, 1984, 26, 355-372; Clowes et al., Laboratory Investigations,1978, 39, 141-150. Moreover, excessive proliferation of airway smoothmuscle cells leads to the development of COPD in the setting of asthmaand chronic bronchitis. Inhibitors of PI3-kinase activity therefore maybe used to prevent vascular restenosis, atherosclerosis, and COPD.

PI3-kinase also plays an important role in leukocyte function (Fuller etal., The Journal of Immunology, 1999, 162(11), 6337-6340; Eder et al.,The Journal of Biological Chemistry, 1998, 273(43), 28025-31) andlymphocyte function (Vicente-Manzanares et al., The Journal ofImmunology, 1999, 163(7), 4001-4012). For example, leukocyte adhesion toinflamed endothelium involves activation of endogenous leukocyteintegrins by a PI3-kinase-dependent signalling process. Furthermore,oxidative burst (Nishioka et al., FEBS Letters, 1998, 441(1), 63-66 andCondliffe, A. M., et al., Blood 2005, 106(4), 1432-40) and cytoskeletalreorganization (Kirsch et al., Proceedings National Academy of SciencesUSA, 1999, 96(11), 6211-6216) in neutrophils appears to involvePI3-kinase signalling. Neutrophil migration and directional movement arealso dependent on PI3-kinase activity (Camps, M., et al., Nat Med, 2005,11(9), 936-43 and Sadhu, C. et al., J Immunol, 2003, 170(5), 2647-54).Thus, inhibitors of PI3-kinase may be useful in reducing leukocyteadhesion and activation at sites of inflammation and therefore may beused to treat acute and/or chronic inflammatory disorders. PI3-kinasealso plays an important role in lymphocyte proliferation and activation,Fruman et al., Science, 1999, 283 (5400), 393-397. In particular, PI3K-δis essential for B cell development and function, including IgM-specificantibody-induced B-cell proliferation (Okkenhaug K et al., Science,2002, 297(5583), 1031-1034), B-cell-receptor-induced DNA synthesis andproliferation, and IL-4-induced survival (Bilancio A et al., Blood,2006, 107, 642-650). These observations indicate that PI3K-δ has acrucial and non-redundant role in B-cell function that is notcompensated by other class I PI3Ks. Given the important role oflymphocytes in auto-immune diseases, an inhibitor of PI3-kinase activitymay be used in the treatment of such disorders (Rommel C, Camps M and JiH, Nat Rev Immunol, 2007, 1038, 191-201).

The anti-cancer treatment defined hereinbefore may be applied as a soletherapy or may involve, in addition to the compound of the invention,conventional surgery or radiotherapy or chemotherapy. Such chemotherapymay include one or more of the following categories of anti-tumouragents:—

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, and hydroxyurea); antitumourantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere and polokinase inhibitors); andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin);(ii) antihormonal agents such as antioestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5α-reductase suchas finasteride;(iii) inhibitors of growth factor function and their downstreamsignalling pathways: included are Ab modulators of any growth factor orgrowth factor receptor targets, reviewed by Stem et al. Critical Reviewsin Oncology/Haematology, 2005, 54, pp 11-29); also included are smallmolecule inhibitors of such targets, for example kinaseinhibitors—examples include the anti-erbB2 antibody trastuzumab[Herceptin™], the anti-EGFR antibody panitumumab, the anti-EGFR antibodycetuximab [Erbitux, C225] and tyrosine kinase inhibitors includinginhibitors of the erbB receptor family, such as epidermal growth factorfamily receptor (EGFR/erbB1) tyrosine kinase inhibitors such asgefitinib or erlotinib, erbB2 tyrosine kinase inhibitors such aslapatinib, and mixed erb1/2 inhibitors such as afatanib; similarstrategies are available for other classes of growth factors and theirreceptors, for example inhibitors of the hepatocyte growth factor familyor their receptors including c-met and ron; inhibitors of the insulinand insulin growth factor family or their receptors (IGFR, IR)inhibitors of the platelet-derived growth factor family or theirreceptors (PDGFR), and inhibitors of signalling mediated by otherreceptor tyrosine kinases such as c-kit, AnLK, and CSF-1R;also included are modulators which target signalling proteins in thewider PI3-kinase signalling pathway, for example, inhibitors of otherPI3-kinase isoforms such as PI3K-β, and ser/thr kinases such as AKT,mTOR, PDK, SGK, PI4K or PIP5K;also included are inhibitors of serine/threonine kinases not listedabove, for example raf inhibitors such as vemurafenib, MEK inhibitorssuch as selumetinib (AZD6244), Abl inhibitors such as imatinib ornilotinib, Btk inhibitors such as ibrutinib, Syk inhibitors such asfostamatinib, aurora kinase inhibitors (for example AZD1152), inhibitorsof other ser/thr kinases such as JAKs, STATs and IRAK4, and cyclindependent kinase inhibitors;iv) modulators of DNA damage signalling pathways, for example PARPinhibitors (e.g. Olaparib), ATR inhibitors or ATM inhibitors;v) modulators of apoptotic and cell death pathways such as Bcl familymodulators (e.g. ABT-263/Navitoclax, ABT-199);

-   -   (vi) antiangiogenic agents such as those which inhibit the        effects of vascular endothelial growth factor, [for example the        anti-vascular endothelial cell growth factor antibody        bevacizumab (Avastin™) and for example, a VEGF receptor tyrosine        kinase inhibitor such as sorafenib, axitinib, pazopanib,        sunitinib and vandetanib (and compounds that work by other        mechanisms (for example linomide, inhibitors of integrin αvβ3        function and angiostatin)];        (vii) vascular damaging agents, such as Combretastatin A4;        (viii) anti-invasion agents, for example c-Src kinase family        inhibitors like (dasatinib, J. Med. Chem., 2004, 47, 6658-6661)        and bosutinib (SKI-606), and metalloproteinase inhibitors like        marimastat, inhibitors of urokinase plasminogen activator        receptor function or antibodies to Heparanase];        (ix) immunotherapy approaches, including for example ex-vivo and        in-vivo approaches to increase the immunogenicity of patient        tumour cells, such as transfection with cytokines such as        interleukin 2, interleukin 4 or granulocyte-macrophage colony        stimulating factor, approaches to decrease T-cell anergy,        approaches using transfected immune cells such as        cytokine-transfected dendritic cells, approaches using        cytokine-transfected tumour cell lines and approaches using        anti-idiotypic antibodies. Specific examples include monoclonal        antibodies targeting PD-1 (e.g. BMS-936558) or CTLA4 (e.g.        ipilimumab and tremelimumab);        (x) Antisense or RNAi based therapies, for example those which        are directed to the targets listed.        (xi) gene therapy approaches, including for example approaches        to replace aberrant genes such as aberrant p53 or aberrant BRCA1        or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy)        approaches such as those using cytosine deaminase, thymidine        kinase or a bacterial nitroreductase enzyme and approaches to        increase patient tolerance to chemotherapy or radiotherapy such        as multi-drug resistance gene therapy.

According to this aspect of the invention there is provided acombination suitable for use in the treatment of cancer comprising acompound of Formula (I) as defined hereinbefore or a pharmaceuticallyacceptable salt thereof and another anti-tumour agent, in particular anyone of the anti tumour agents listed under (i)-(xi) above. Inparticular, the anti-tumour agent listed under (i)-(xi) above is thestandard of care for the specific cancer to be treated; the personskilled in the art will understand the meaning of “standard of care”.

Therefore in a further aspect of the invention there is provided acompound of Formula (I) or a pharmaceutically acceptable salt thereof incombination with another anti-tumour agent, in particular an anti-tumouragent selected from one listed under (i)-(xi) herein above.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith another anti-tumour agent, in particular an anti-tumour agentselected from one listed under (i) above.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and any one of the anti tumour agents listed under (i)above.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and a taxoid, such as for example taxol or taxotere,conveniently taxotere.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith another anti-tumour agent, in particular an anti-tumour agentselected from one listed under (ii) herein above.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and any one of antihormonal agents listed under (ii) above,for example any one of the anti-oestrogens listed in (ii) above.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith an mTOR inhibitor, such as those disclosed in WO2008/023161, forexample

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and an mTOR inhibitor, such as those disclosed inWO2008/023161, for example

In particular, the mTOR inhibitor is AZD2014, which has the followingstructure:

In one aspect, the above combination of the compound of formula (I) andAZD2014 is suitable for use in the treatment of ER+ve breast cancer,optionally in combination with standard of care hormonal therapy.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith an inhibitor of PI3K-β.

The combination of a compound of formula (I) with an inhibitor of PI3K-βmay be particularly useful in the treatment of tumours, for example,prostate, breast (for example triple negative breast), squamous cellNSCLC and renal cancer, with a background of PTEN loss.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and an inhibitor of PI3K-β.

In one aspect, the inhibitors of PI3K-β described herein also have somePI3K-δ inhibitory activity.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith an inhibitor of PI3K-β, such as any one of the examples inInternational patent application WO2011/051704.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and an inhibitor of PI3K-β, such as anyone of the examplesin International patent application WO2011/051704.

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith an inhibitor of PI3K-β and PI3K-δ, such as8-((1R)-1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide(example 3.06b in International patent application WO2011/051704, alsoknown as AZD8186) or a pharmaceutically-acceptable salt thereof:

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and an inhibitor of PI3K-β and PI3K-δ, such as8-((1R)-1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide(example 3.06b in International patent application WO2011/051704, alsoknown as AZD8186) or a pharmaceutically-acceptable salt thereof:

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith an inhibitor of AKT kinase, such as(S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide(AZD5363) or a pharmaceutically-acceptable salt thereof (see for exampleWO2009/047563).

The combination of a compound of Formula (I) and an AKT inhibitor may beparticularly useful in treating tumours with a higher prevalence ofmutation in PIK3CA gene, such as ER+ve breast cancer, endometrial,ovarian, squamous cell NSCLC, gastric, bladder and biliary tract cancer.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and an inhibitor of AKT kinase, such as(S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide(AZD5363) or a pharmaceutically-acceptable salt thereof (see for exampleWO2009/047563).

In a further aspect of the invention there is provided a compound ofFormula (I) or a pharmaceutically acceptable salt thereof in combinationwith olaparib(4-[3-(4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one)or a pharmaceutically-acceptable salt thereof.

The combination of a compound of Formula (I) and olaparib may beparticularly useful both in triple negative breast cancer, either BRCAwild type or deficient, and in estrogen receptor positive (ER+ve) breastcancer, particularly those with mutations in the PIK3CA gene.

In a further aspect of the invention there is provided a combinationsuitable for use in the treatment of cancer comprising a compound ofFormula (I) as defined hereinbefore or a pharmaceutically acceptablesalt thereof and olaparib(4-[3-(4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one)or a pharmaceutically-acceptable salt thereof.

Particular combinations of the invention comprise any one of thecompounds of the Examples herein (or a pharmaceutically acceptable saltthereof) and an mTOR inhibitor, PI3Kβ inhibitor, inhibitor of AKT kinaseor olaparib as described hereinabove. Further particular combinations ofthe invention comprise Example 3 (or a pharmaceutically acceptable saltthereof) and an mTOR inhibitor, PI3Kβ inhibitor, inhibitor of AKT kinaseor olaparib as described hereinabove. Further particular combinations ofthe invention comprise Example 3 (or a pharmaceutically acceptable saltthereof) and a PI3Kβ inhibitor, inhibitor of AKT kinase or olaparib (ora pharmaceutically-acceptable salt of any one of these three), asdescribed hereinabove. Still further particular examples of combinationsof the invention comprise Example 3 (or a pharmaceutically acceptablesalt thereof) and any one of AZD8186, AZD5363 and olaparib (or apharmaceutically-acceptable salt of any one of these three). Anotherexample of a combination of the invention comprises Example 3 andAZD2014.

In all of the above combinations, it will be understood that thecombination may also be dosed with standard of care treatment, asunderstood by the skilled person, such as other treatments from (i) to(xi) hereinbefore. For example, when it is intended to use any of theabove combinations for the treatment of ER+ve breast cancer, standard ofcare hormonal therapy (such as those agents listed under (ii) above) maybe used in conjunction with the combination of the invention. In otheraspects, suitably the standard of care may be selected from (i) above.

Therefore in a further aspect of the invention, there is provided atriple combination suitable for use in the treatment of cancer

-   -   a) a compound of formula (I) (such as Example 3) or a        pharmaceutically-acceptable salt thereof;    -   b) an mTOR inhibitor, PI3Kβ inhibitor, inhibitor of AKT kinase        or olaparib or a pharmaceutically-acceptable salt thereof; and    -   c) standard of care therapy for the cancer to be treated.

Suitably standard of care therapy may be dosed according to its usualdosing regimen, as understood by the skilled person.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises Example 3 or apharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises Example 3 or apharmaceutically acceptable salt thereof in combination with AZD5363,AZD8186 or olaparib, (or a pharmaceutically-acceptable salt of any oneof these three) in association with a pharmaceutically acceptablediluent or carrier.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises a compound of Formula (I) ora pharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier foruse in treating cancer.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises Example 3 or apharmaceutically acceptable salt thereof in combination with ananti-tumour agent selected from one listed under (i)-(xi) herein above,in association with a pharmaceutically acceptable diluent or carrier foruse in treating cancer.

According to a further aspect of the invention there is provided apharmaceutical composition which comprises Example 3 or apharmaceutically acceptable salt thereof in combination with AZD5363,AZD8186 or olaparib, (or a pharmaceutically-acceptable salt of any oneof these three) in association with a pharmaceutically acceptablediluent or carrier for use in treating cancer.

According to another feature of the invention there is provided the useof a compound of the Formula (I) or a pharmaceutically acceptable saltthereof in combination with an anti-tumour agent selected from onelisted under (i)-(xi) herein above, in the manufacture of a medicamentfor use in cancer in a warm-blooded animal, such as man.

According to another feature of the invention there is provided the useof Example 3 or a pharmaceutically acceptable salt thereof incombination with an anti-tumour agent selected from one listed under(i)-(xi) herein above, in the manufacture of a medicament for use incancer in a warm-blooded animal, such as man.

According to another feature of the invention there is provided the useof Example 3 or a pharmaceutically acceptable salt thereof incombination with AZD5363, AZD8186 or olaparib, (or apharmaceutically-acceptable salt of any one of these three) in themanufacture of a medicament for use in cancer in a warm-blooded animal,such as man.

Therefore in an additional feature of the invention, there is provided amethod of treating cancer in a warm-blooded animal, such as man, in needof such treatment which comprises administering to said animal aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in combination with an anti-tumour agentselected from one listed under (i)-(xi) herein above.

Therefore in an additional feature of the invention, there is provided amethod of treating cancer in a warm-blooded animal, such as man, in needof such treatment which comprises administering to said animal aneffective amount of Example 3 or a pharmaceutically acceptable saltthereof in combination with an anti-tumour agent selected from onelisted under (i)-(xi) herein above.

Therefore in an additional feature of the invention, there is provided amethod of treating cancer in a warm-blooded animal, such as man, in needof such treatment which comprises administering to said animal aneffective amount of Example 3 or a pharmaceutically acceptable saltthereof in combination with AZD5363, AZD8186 or olaparib (or apharmaceutically-acceptable salt of any one of these three).

According to a further aspect of the present invention there is provideda kit comprising a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in combination with an anti-tumour agentselected from one listed under (i)-(xi) herein above.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an anti-tumour agent selected from one listed under (i)-(xi) hereinabove in a second unit dosage form; andc) container means for containing said first and second dosage forms.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an anti-tumour agent selected from one listed under (i)-(xi) hereinabove in a second unit dosage form;c) container means for containing said first and second dosage forms;and optionallyd) instructions for use.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an mTOR inhibitor, such as those disclosed in WO2008/023161, forexample

in a second unit dosage form; andc) container means for containing said first and second dosage forms.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an inhibitor of PI3K-β, such as any one of the examples inInternational patent application WO2011/051704, or a pharmaceuticallyacceptable salt thereof in a second unit dosage form; andc) container means for containing said first and second dosage forms.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an inhibitor of PI3K-β, such as any one of the examples inInternational patent application WO2011/051704, or a pharmaceuticallyacceptable salt thereof in a second unit dosage form;c) container means for containing said first and second dosage forms;and optionallyd) instructions for use.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an inhibitor of PI3K-β and PI3K-δ which is8-((1R)-1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide(example 3.06b in International patent application WO2011/051704, alsoknown as AZD8186), or a pharmaceutically acceptable salt thereof in asecond unit dosage form; andc) container means for containing said first and second dosage forms.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an inhibitor of PI3K-β and PI3K-δ which is8-((1R)-1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide(example 3.06b in International patent application WO2011/051704, alsoknown as AZD8186), or a pharmaceutically acceptable salt thereof in asecond unit dosage form;c) container means for containing said first and second dosage forms;and optionallyd) instructions for use.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an inhibitor of AKT kinase, such as(S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamideor a pharmaceutically-acceptable salt thereof (AZD5363, see for exampleWO2009/047563), in a second unit dosage form;c) container means for containing said first and second dosage forms;and optionallyd) instructions for use.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an inhibitor of AKT kinase, such as(S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamideor a pharmaceutically-acceptable salt thereof (AZD5363, see for exampleWO2009/047563), in a second unit dosage form; andc) container means for containing said first and second dosage forms.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) an inhibitor of AKT kinase, such as(S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamideor a pharmaceutically-acceptable salt thereof (AZD5363, see for exampleWO2009/047563), in a second unit dosage form;c) container means for containing said first and second dosage forms;and optionallyd) instructions for use.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) olaparib(4-[3-(4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one)or a pharmaceutically acceptable salt thereof, in a second unit dosageform; andc) container means for containing said first and second dosage forms.

According to a further aspect of the present invention there is provideda kit comprising:

a) a compound of Formula (I) or a pharmaceutically acceptable saltthereof in a first unit dosage form;b) olaparib(4-[3-(4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one)or a pharmaceutically acceptable salt thereof, in a second unit dosageform;c) container means for containing said first and second dosage forms;and optionallyd) instructions for use.

In all of the above combinations, uses, methods of treatment and kits,AZD5363, AZD8186 and olaparib may be in the form of free bases or in theform of a pharmaceutically-acceptable salt. Therefore in one embodimentAZD5363 is in the form of a free base; in a further embodiment AZD5363is in the form of a pharmaceutically-acceptable salt. In anotherembodiment AZD8186 is in the form of a free base; in a furtherembodiment AZD8186 is in the form of a pharmaceutically-acceptable salt.In another embodiment olaparib is in the form of a free base; in afurther embodiment olaparib is in the form of apharmaceutically-acceptable salt.

Although the compounds of the Formula (I) are primarily of value astherapeutic agents for use in warm-blooded animals (including man), theyare also useful whenever it is required to inhibit the effects ofPI3-kinase-α and -δ. Thus, they are useful as pharmacological standardsfor use in the development of new biological tests and in the search fornew pharmacological agents.

Herein, where the term “combination” is used it is to be understood thatthis refers to simultaneous, separate or sequential administration. Inone aspect of the invention “combination” refers to simultaneousadministration. In another aspect of the invention “combination” refersto separate administration. In a further aspect of the invention“combination” refers to sequential administration. Where theadministration is sequential or separate, the delay in administering thesecond component should not be such as to lose the beneficial effect.

In one embodiment sequential treatment involves administration of eachcomponent of the combination within a period of 11 days. In anotherembodiment this period is 10 days. In another embodiment this period is9 days. In another embodiment this period is 8 days. In anotherembodiment this period is 7 days. In another embodiment this period iswithin 6 days. In another embodiment this period is within 5 days. Inanother embodiment this period is within 4 days. In another embodimentthis period is within 3 days. In another embodiment this period iswithin 2 days. In another embodiment this period is within 24 hours. Inanother embodiment this period is within 12 hours.

Sequential and co-administration are both exemplified herein in thecombination experiments with Example 3 and AZD5363 in BT474 model. Inthis example, sequential administration is illustrated by dosing AZD5363for 2 days followed by Example 3 for 2 days, then 3 days with no dose ofeither agent before the pattern is repeated (“dosage cycle”).Co-administration is illustrated with a dosing regimen where bothAZD5363 and Example 3 are dosed for 2 days, followed by 5 days with nodose of either agent. In these two examples, sequential administrationappears to be more effective in causing tumour regression, illustratingthe potential importance of optimising the regimen. Further potentialco-dosing regimens include:

1) a dosage cycle where both AZD5363 and Example 3 are dosed for 2 days,followed by 3 days with no dose of either agent;2) a dosage cycle where both AZD5363 and Example 3 are dosed for 3 days,followed by 4 days with no dose of either agent;3) a dosage cycle where both AZD5363 and Example 3 are dosed for 4 days,followed by 3 days with no dose of either agent;4) a dosage cycle where both AZD5363 and Example 3 are dosed for 5 days,followed by 2 days with no dose of either agent5) a dosage cycle where AZD5363 and Example 3 are dosed every other day6) a dosage cycle where AZD5363 and Example 3 are dosed every three days7) a dosage cycle where AZD5363 and Example 3 on dosed on a weeklyschedule with 3 and 4 day gaps between dosing (e.g. Monday/Thursday)8) a dosage cycle where AZD5363 and Example 3 are dosed on a weeklyschedule with 2 and 3 days gaps between dosing (e.g.Monday/Wednesday/Friday)

Combinations of compounds of formula (I), particularly Example 3, withan mTOR inhibitor, such as AZD2014 or a PI3K-β inhibitor (such as theβ/δ inhibitor AZD8186) may suitably be dosed in a similar regimen tothose described above for the combination of Example 3 and AZD5363.

A combination of a compound of formula (I) and olaparib may be dosedaccording to a regimen where olaparib is dosed daily and the compound offormula (I) is dosed according to an intermittent dosing schedule (suchas for example 2 days dosing followed by 3 to 5 days with no dose).

Each of these illustrative dosing regimes comprise a further aspect ofthe invention. Each of these illustrative dosing regimes may also beapplied to combinations with other anti-tumour agents listed in (i) to(xi) above.

It may be advantageous, within a given dosage cycle, to administer onespecific component of the combination before the other—i.e. sequentialdosing.

Therefore, in one embodiment the sequential administration comprises thesequential administration of the compound of formula (I) (particularlyExample 3) prior to the administration of the other anti-tumour agentlisted in (i) to (xi) above, particularly an anti-tumour agent selectedfrom AZD5363, AZD8186 and olaparib, within a dosage cycle.

In another embodiment the sequential administration comprises thesequential administration of the anti-tumour agent listed in (i) to (xi)above, particularly an anti-tumour agent selected from AZD5363, AZD8186and olaparib, prior to the administration of compound of formula (I)(particularly Example 3) within a dosage cycle.

In one embodiment, the anti-tumour agent listed in (i) to (xi) above andthe compound of formula (I) are dosed up to 2 days apart. In anotherembodiment, the anti-tumour agent listed in (i) to (xi) above and thecompound of formula (I) are dosed up to 1 day apart. In anotherembodiment, the anti-tumour agent listed in (i) to (xi) above and thecompound of formula (I) are dosed up to 18 hours apart. In anotherembodiment, the anti-tumour agent listed in (i) to (xi) above and thecompound of formula (I) are dosed up to 12 hours apart. In anotherembodiment, the anti-tumour agent listed in (i) to (xi) above and thecompound of formula (I) are dosed up to 6 hours apart. In anotherembodiment, the anti-tumour agent listed in (i) to (xi) above and thecompound of formula (I) are dosed up to 3 hours apart.

In further embodiments the dosage cycle may be from 5 to 10 days inlength.

In further embodiments the dosage cycle may be from 6 to 10 days inlength.

In further embodiments the dosage cycle may be from 7 to 9 days inlength.

In further embodiments the dosage cycle may be from 6 to 8 days inlength.

In further embodiments the dosage cycle may be 10 days in length.

In further embodiments the dosage cycle may be 9 days in length.

In further embodiments the dosage cycle may be 8 days in length.

In further embodiments the dosage cycle may be 7 days in length.

In further embodiments the dosage cycle may be 6 days in length.

In further embodiments the dosage cycle may be 5 days in length.

In further embodiments the dosage cycle may involve the compound offormula (I) (particularly Example 3) being dosed for 2-4 consecutivedays and not being dosed for the other days within a dosage cycle of 6to 9 days in length.

In further embodiments the dosage cycle may involve the compound offormula (I) (particularly Example 3) being dosed for 3-4 consecutivedays and not being dosed for the other days within a dosage cycle of 6to 9 days in length; (for example, 7 days in length).

In further embodiments the dosage cycle may involve the compound offormula (I) (particularly Example 3) being dosed for 3-5 consecutivedays and not being dosed for the other days within a dosage cycle of 7to 10 days in length.

In further embodiments the dosage cycle may involve the compound offormula (I) (particularly Example 3) being dosed for 5 consecutive daysand not being dosed for the other days within a dosage cycle of 6 to 9days in length.

In further embodiments the dosage cycle may involve the compound offormula (I) (particularly Example 3) being dosed for 4 consecutive daysand not being dosed for the other days within a dosage cycle of 6 to 9days in length; (for example, 7 days in length).

In further embodiments the dosage cycle may involve the compound offormula (I) (particularly Example 3) being dosed for 3 consecutive daysand not being dosed for the other days within a dosage cycle of 6 to 9days in length.

Dosage cycles may be separated by a number of days where none of theactive combination components are administered.

Combination therapy as described above may be added on top of standardof care therapy typically carried out according to its usual prescribingdosage schedule.

Personalised Healthcare

Another aspect of the present invention is based on identifying a linkbetween the status of the gene encoding phosphoinositide-3-kinase,catalytic, alpha polypeptide (PIK3CA) and susceptibility to treatmentwith a compound of Formula (I). This therefore provides opportunities,methods and tools for selecting patients for treatment with a compoundof Formula (I), particularly cancer patients, and/or avoiding treatmentof patients less likely to respond therapeutically to the treatment thusavoiding unnecessary treatment and any side effects that may beassociated with such ineffective treatment.

The present invention relates to patient selection tools and methods(including personalised medicine). The selection is based on whether thetumour cells to be treated possess wild-type or mutant PIK3CA gene. ThePIK3CA gene status can therefore be used as a biomarker ofsusceptibility to treatment with a PI3K-α and -δ inhibitor.

There is a clear need for biomarkers that will enrich for or selectpatients whose tumours will respond to treatment with a PI3K-α and -δinhibitor, such as a compound of Formula (I). Patient selectionbiomarkers that identify the patients most likely to respond to an agentare ideal in the treatment of cancer, since they reduce the unnecessarytreatment of patients with non-responding tumours to the potential sideeffects of such agents.

A biomarker can be described as “a characteristic that is objectivelymeasured and evaluated as an indicator of normal biologic processes,pathogenic processes, or pharmacologic responses to a therapeuticintervention”. A biomarker is any identifiable and measurable indicatorassociated with a particular condition or disease where there is acorrelation between the presence or level of the biomarker and someaspect of the condition or disease (including the presence of, the levelor changing level of, the type of, the stage of, the susceptibility tothe condition or disease, or the responsiveness to a drug used fortreating the condition or disease). The correlation may be qualitative,quantitative, or both qualitative and quantitative. Typically abiomarker is a compound, compound fragment or group of compounds. Suchcompounds may be any compounds found in or produced by an organism,including proteins (and peptides), nucleic acids and other compounds.

Biomarkers may have a predictive power, and as such may be used topredict or detect the presence, level, type or stage of particularconditions or diseases (including the presence or level of particularmicroorganisms or toxins), the susceptibility (including geneticsusceptibility) to particular conditions or diseases, or the response toparticular treatments (including drug treatments). It is thought thatbiomarkers will play an increasingly important role in the future ofdrug discovery and development, by improving the efficiency of researchand development programs. Biomarkers can be used as diagnostic agents,monitors of disease progression, monitors of treatment and predictors ofclinical outcome. For example, various biomarker research projects areattempting to identify markers of specific cancers and of specificcardiovascular and immunological diseases. It is believed that thedevelopment of new validated biomarkers will lead both to significantreductions in healthcare and drug development costs and to significantimprovements in treatment for a wide variety of diseases and conditions.

In order to optimally design clinical trials and to gain the mostinformation from these trials, a biomarker may be required. The markermay be measurable in surrogate and tumour tissues. Ideally these markerswill also correlate with efficacy and thus could ultimately be used forpatient selection.

Thus, the technical problem underlying this aspect of the presentinvention is the identification of means for stratification of patientsfor treatment with a compound of Formula (I). The technical problem issolved by provision of the embodiments characterized in the claimsand/or description herein.

As detailed in the examples herein, it was found that cells that possessa mutation in the PIK3CA are generally more susceptible to growthinhibition by the compound of Formula (I).

The invention provides a method of determining sensitivity of cells to acompound of Formula (I). The method comprises determining the status ofPIK3CA gene in said cells. The cells are identified as likely to besensitive to a compound of Formula I if the cells possess a mutatedPIK3CA gene. Those patients with a mutated PIK3CA gene are thereforepredicted to be particularly susceptible to treatment with a compound ofFormula (I). A cell is defined as sensitive to a compound of Formula (I)if it inhibits the increase in cell number in a cell growth assay(either through inhibition of cell proliferation and/or throughincreased cell death). Methods of the invention are useful forpredicting which cells are more likely to respond to a compound ofFormula (I) by growth inhibition.

The present invention is further based, in part, on methods that can beused to determine a patient's responsiveness to a compound of Formula(I) including determining whether to administer a compound of Formula(I). Specifically the methods of the present invention include thedetermination of the gene status of PIK3CA. The presence of a mutatedPIK3CA gene indicates that the tumour cells are more likely to respondby growth inhibition when contacted with a compound of Formula (I). ThePIK3CA gene status can therefore be used to select patients fortreatment with a compound of Formula (I).

Furthermore an in vitro method for the identification of a patientlikely to be sensitive to a compound of Formula (I) is disclosed. Alsodisclosed are uses of an oligo- or polynucleotide primers or probescapable of detecting the mutation status of PIK3CA gene is provided.Also disclosed are use of ‘kits for the detection of PIK3CA mutations,including but not limited to, the PIK3CA mutation detection kitsmarketed by diagnostic companies including Qiagen and Roche MolecularSystems. In another embodiment, the invention pertains to an in vitromethod for determining whether a patient suffering from cancer is likelyto be a responder to a pharmaceutical treatment with a compound ofFormula (I), said method comprising the steps of: (i) obtaining a samplerepresentative of the tumour that was previously collected from saidpatient; and, (ii) determining whether the PIK3CA genes contain amutation in said sample. A mutation in PIK3CA gene is indicative ofincreased likelihood of a response to treatment with a compound ofFormula (I). As a single gene biomarker test, identification of tumoursthat contain a PIK3CA mutation will enrich for response to a compound ofFormula (I). Individual tumours that contain a PIK3CA mutation have thegreatest likelihood of responding to a compound of Formula (I).

A sample “representative of the tumour” can be the actual tumour sampleisolated, or may be a sample that has been further processed, e.g. asample of PCR amplified nucleic acid from the tumour sample.

DEFINITIONS

In this Personalised Healthcare section:

“Allele” refers to a particular form of a genetic locus, distinguishedfrom other forms by its particular nucleotide or amino acid sequence.

“Amplification reactions” are nucleic acid reactions which result inspecific amplification of target nucleic acids over non-target nucleicacids. The polymerase chain reaction (PCR) is a well known amplificationreaction.

“Cancer” is used herein to refer to neoplastic growth arising fromcellular transformation to a neoplastic phenotype. Such cellulartransformation often involves genetic mutation.

“Gene” is a segment of DNA that contains all the information for theregulated biosynthesis of an RNA product, including a promoter, exons,introns, and other sequence elements which may be located within 5′ or3′ flanking regions (not within the transcribed portions of the gene)that control expression.

“Gene status” refers to whether the gene is wild type or not (i.e.mutant).

“Label” refers to a composition capable of producing a detectable signalindicative of the presence of the target polynucleotide in an assaysample. Suitable labels include radioisotopes, nucleotide chromophores,enzymes, substrates, fluorescent molecules, chemiluminescent moieties,magnetic particles, bioluminescent moieties, and the like. As such, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.

“Non-synonymous variation” refers to a variation (variance) in oroverlapping the coding sequence of a gene that result in the productionof a distinct (altered) polypeptide sequence. These variations may ormay not affect protein function and include missense variants (resultingin substitution of one amino acid for another), nonsense variants(resulting in a truncated polypeptide due to generation of a prematurestop codon) and insertion/deletion variants.

“Synonymous variation” refers to a variation (variance) in the codingsequence of a gene that does not affect sequence of the encodedpolypeptide. These variations may affect protein function indirectly(for example by altering expression of the gene), but, in the absence ofevidence to the contrary, are generally assumed to be innocuous.

“Nucleic acid” refers to single stranded or double stranded DNA and RNAmolecules including natural nucleic acids found in nature and/ormodified, artificial nucleic acids having modified backbones or bases,as are known in the art.

“Primer” refers to a single stranded DNA oligonucleotide sequencecapable of acting as a point of initiation for synthesis of a primerextension product which is complementary to the nucleic acid strand tobe copied. The length and sequence of the primer must be such that theyare able to prime the synthesis of extension products. A typical primercontains at least about 7 nucleotides in length of a sequencesubstantially complementary to the target sequence, but somewhat longerprimers are preferred. Usually primers contain about 15-26 nucleotides,but longer or shorter primers may also be employed.

“Polymorphic site” is a position within a locus at which at least twoalternative sequences are found in a population.

“Polymorphism” refers to the sequence variation observed in anindividual at a polymorphic site. Polymorphisms include nucleotidesubstitutions, insertions, deletions and microsatellites and may, butneed not, result in detectable differences in gene expression or proteinfunction. In the absence of evidence of an effect on expression orprotein function, common polymorphisms, including non-synonomousvariants, are generally considered to be included in the definition ofwild-type gene sequence. A catalog of human polymorphisms and associatedannotation, including validation, observed frequencies, and diseaseassociation, is maintained by NCBI (dbSNP:http://www.ncbi.nlm.nih.gov/projects/SNP/). Please note that the term“polymorphism” when used in the context of gene sequences should not beconfused with the term “polymorphism” when used in the context of solidstate form of a compound, that is the crystalline or amorphous nature ofa compound. The skilled person will understand the intended meaning byits context.

“Probe” refers to single stranded sequence-specific oligonucleotideswhich have a sequence that is exactly complementary to the targetsequence of the allele to be detected.

“Response” is defined by measurements taken according to ResponseEvaluation Criteria in Solid Tumours (RECIST) involving theclassification of patients into two main groups: those that show apartial response or stable disease and those that show signs ofprogressive disease.

“Stringent hybridisation conditions” refers to an overnight incubationat 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulphate, and 20 pg/mI denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

“Survival” encompasses a patients' overall survival and progression-freesurvival.

“Overall survival” (OS) is defined as the time from the initiation ofdrug administration to death from any cause. “Progression-free survival”(PFS) is defined as the time from the initiation of drug administrationto first appearance of progressive disease or death from any cause.

According to one aspect of the invention there is provided a method forselecting a patient for treatment with a compound of Formula (I), themethod comprising providing a tumour cell containing sample from apatient; determining whether the PIK3CA gene in the patient's tumourcell containing sample is wild type or mutant; and selecting a patientfor treatment with a compound of Formula (I) based thereon.

The method may include or exclude the actual patient sample isolationstep. Thus, according to one aspect of the invention there is provided amethod for selecting a patient for treatment with a compound of Formula(I), the method comprising determining whether the PIK3CA gene in atumour cell containing sample previously isolated from the patient iswild type or mutant; and selecting a patient for treatment with acompound of Formula (I) based thereon.

In one embodiment, the patient is selected for treatment with a compoundof Formula (I) if the tumour cell DNA has a mutant PIK3CA gene. In otherembodiments, a patient whose tumour cell DNA possesses a wild typePIK3CA gene is not selected for treatment with a compound of Formula(I).

According to another aspect of the invention there is provided a methodfor predicting a patient's responsiveness to a compound of Formula (I),the method comprising determining whether the PIK3CA gene in thepatient's tumour cells is wild type or mutant and based thereon,predicting a patient's responsiveness to treatment with a compound ofFormula (I).

According to another aspect of the invention there is provided a methodfor determining the likelihood of effectiveness of treatment with acompound of formula I in a human patient affected with cancercomprising: determining whether the PIK3CA gene(s) in the patient'stumour cells is wild type or mutant and based thereon, predicting apatient's responsiveness to treatment with a compound of Formula (I).

For the purpose of this invention, a gene status of wild-type is meantto indicate normal or appropriate expression of the gene and normalfunction of the encoded protein. In contrast, mutant status is meant toindicate abnormal or inappropriate gene expression, or expression of aprotein with altered function, consistent with the known roles of mutantPIK3CA in cancer (as described herein). Any number of genetic orepigenetic alterations, including but not limited to mutation,amplification, deletion, genomic rearrangement, or changes inmethylation profile, may result in a mutant status. However, if suchalterations nevertheless result in appropriate expression of the normalprotein, or a functionally equivalent variant, then the gene status isregarded as wild-type. Examples of variants that typically would notresult in a functional mutant gene status include synonomous codingvariants and common polymorphisms (synonymous or non-synonymous). Asdiscussed below, gene status can be assessed by a functional assay, orit may be inferred from the nature of detected deviations from areference sequence.

In certain embodiments the wild-type or mutant status of the PIK3CA geneis determined by the presence or absence of non-synonymous nucleic acidvariations in the genes. Observed non-synonymous variationscorresponding to known common polymorphisms with no annotated functionaleffects do not contribute to a gene status of mutant.

Other variations in the PIK3CA gene that signify mutant status includesplice site variations that decrease recognition of an intron/exonjunction during processing of pre-mRNA to mRNA. This can result in exonskipping or the inclusion of normally intronic sequence in spliced mRNA(intron retention or utilization of cryptic splice junctions). This can,in turn, result in the production of aberrant protein with insertionsand/or deletions relative to the normal protein. Thus, in otherembodiments, the gene has a mutant status if there is a variant thatalters splice site recognition sequence at an intron/exon junction.

In addition, the measurement of mutation status or activation status ofadditional genes, such as Kras, a potential marker of resistance intumours with aberrant or deregulated PIK3CA or PI3K-α, could helpincrease the predictivity of a Personalised Medicine approach.

In a survey we conducted at AstraZeneca on breast cancers (based onCOSMIC database (Welcome Trust Sanger Institute, September 2011), >55different mutations in the PIK3CA gene were identified from across adataset covering >5K human tumours. The majority of mutations occurredat <1% frequency, 3 occurred at 1-3% frequency, but 4 mutationsaccounted for ˜88% of total PIK3CA mutations. These were kinase domainmissense mutations in the C terminal kinase domain, H1047R (55%) andH1047L (5%), and the helical domain residues, E545K (18%) and E542K(11%). A longer list of other prevalent breast cancer mutations,although not intended to be exhaustive, encompasses R38H, R38C, R88Q,N345K, C420R, E453Q, P539R, E542K, E545K, E545A, Q546K, Q546P, M1043I,M1043V, H1047R, H1047L, H1047Y. Hence diagnostic assays can be builtthat focus on detection of the most common mutations, thereby allowingidentification of the majority of PIK3CA mutations. For example theCobas™ PIK3CA Mutation Test from Roche Molecular Systems is designed todetect 17 mutations in exons 1, 4, 7, 9 and 20 of the PIK3CA gene(E542K, E545A, E545G, E545K, E545D, Q546K, Q546R, Q546E, Q546L, N345K,C420R, R88Q, H1047L, H1047R, H1047Y, G1049R and M1043I) in DNA isolatedfrom formalin-fixed paraffin-embedded tumour samples. This kit iscapable of picking up to ˜95% of mutations in ER+ve breast cancer. Thedistribution of mutations differs across other tumour types and thediagnostic strategy may be adapted accordingly. For example, inendometrial cancer, there is a more even distribution of mutationsspread throughout the PIK3CA gene coding sequence and with larger numberof mutations in the N terminal region of the protein (communicated byDouglas A. Levine, M. D, TCGA 2nd Annual Symposium, Nov. 28, 2012),compared with breast cancers.

For PIK3CA, reference sequences are available for the gene (GenBankaccession number: NG_012113), mRNA (GenBank accession number:NM_006218), and protein (GenBank accession number: NP_006209 orSwiss-Prot accession: P42336). The reference gene (genomic region)sequences include 5000 bases of upstream sequence and 2000 bases ofdownstream sequence. Mutations within PIK3CA are well known (COSMICdatabase—Welcome Trust Sanger Institute), and the person of skill in theart will be able to determine the PIK3CA gene status, i.e. whether aparticular PIK3CA gene is wild type or mutant, based on comparison ofDNA or protein sequence with wild type.

It will be apparent that the gene and mRNA sequences disclosed forPIK3CA and the p110α catalytic subunit of PI3-kinase alpha proteinsequence are each a representative sequence. In normal individuals thereare two copies of each gene, a maternal and paternal copy, which willlikely have some sequence differences, moreover within a populationthere will exist numerous allelic variants of the gene sequence. Othersequences regarded as wild type include those that possess one or moresynonymous changes to the nucleic acid sequence (which changes do notalter the encoded protein sequence), non-synonymous common polymorphisms(e.g. germ-line polymorphisms) which alter the protein sequence but donot affect protein function, and intronic non-splice-site sequencechanges.

According to another aspect of the invention there is provided a methodfor determining the likelihood of effectiveness of treatment with acompound of Formula (I) in a human patient affected with cancercomprising: detecting the presence or absence of at least onenon-synonymous nucleic acid variance in the PIK3CA gene of said patientrelative to the wild type gene, wherein the presence of at least onesomatic non-synonymous nucleic acid variance in the PIK3CA geneindicates that treatment with the compound of Formula (I) is likely tobe effective.

According to another aspect of the invention there is provided a methodfor assessing the susceptibility of an individual to treatment with acompound of Formula (I), which method comprises:

-   -   (i) determining the non-synonymous mutation status of the PIK3CA        gene in tumour cell DNA from the individual; and,    -   (ii) determining the likely susceptibility of the individual to        treatment with a compound of Formula (I) by reference to the        non-synonymous mutation status of the PIK3CA gene in the tumour        cells.

There are numerous techniques available to the person skilled in the artto determine the gene status of PIK3CA. The gene status can bedetermined by determination of the nucleic acid sequence. This could bevia direct sequencing of the full-length gene or analysis of specificsites within the gene, e.g. commonly mutated sites.

An alternative means for determining whether or not the PIK3CA gene iswild type or mutant is to assess the function of the transcribed gene.Functional mutation of this PIK3CA gene produces a protein that hasincreased lipid kinase activity resulting in increased downstreamsignalling of the pathway in cells, including, but not limited to,activation of Akt and S6 kinase. The assays to assess the functionalstatus of PIK3CA variants when expressed in cells include but are notlimited to:

(i) increased production of the product of the kinase activity of thePIK3CA gene, phosphatidylinositol-trisphosphate (PI(3,4,5)P3);(ii) increased levels of phosphorylated Akt or S6 kinase;(iii) increased focus and colony formation of NIH-3T3 cells transfectedwith the variant of PIK3CA; (Ikenoue T et al., Cancer Res., 2005 65,4562-4567).

Samples

The patient's sample to be tested for the gene status can be any tumourtissue or tumour-cell containing sample obtained or obtainable from theindividual. The test sample is conveniently a sample of blood, mouthswab, biopsy, or other body fluid or tissue obtained from an individual.Particular examples include: circulating tumour cells, circulating DNAin the plasma or serum, cells isolated from the ascites fluid of ovariancancer patients, lung sputum for patients with tumours within the lung,a fine needle aspirate from a breast cancer patient, urine, peripheralblood, a cell scraping, a hair follicle, a skin punch or a buccalsample.

It will be appreciated that the test sample may equally be a nucleicacid sequence corresponding to the sequence in the test sample, that isto say that all or a part of the region in the sample nucleic acid mayfirstly be amplified using any convenient technique e.g. polymerasechain reaction (PCR), before analysis. The nucleic acid may be genomicDNA or fractionated or whole cell RNA. In particular embodiments the RNAis whole cell RNA and is used directly as the template for labelling afirst strand cDNA using random primers or poly A primers. The nucleicacid or protein in the test sample may be extracted from the sampleaccording to standard methodologies (see Green & Sambrook, Eds.,Molecular Cloning: A Laboratory Manual, (2012, 4th edition, Vol. 1-3,ISBN 9781936113422), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

The diagnostic methods of the invention can be undertaken using a samplepreviously taken from the individual or patient. Such samples may bepreserved by freezing or fixed and embedded in formalin-paraffin orother media. Alternatively, a fresh tumour cell containing sample may beobtained and used.

The methods of the invention can be applied using cells from any tumour.Suitable tumours for treatment with a compound of Formula (I) have beendescribed hereinbefore.

Mutations in PIK3CA are found broadly in clinical tumours, but theprevalence of mutations in each gene varies significantly by tumourtissue type. For example, PIK3CA mutations are relatively common inbreast cancer but relatively rare in kidney tumours.

TABLE 1 Table 1: Prevalence of PIK3CA mutations in clinical samples.Source for PIK3CA information is the COSMIC database (release v62). Thepatient selection methods of the invention may be particularly useful inthe disease (tissue) segments where there is a high incidence of PIK3CAmutations (e.g. breast, urinary tract, endometrium, large intestine,cervix etc.). PIK3CA mutation Tissue prevalence (%) Penis 29 Endometrium26 Breast 26 Small_intestine 20 Urinary tract 17 Skin 13 Large intestine12 Stomach 9 Biliary_tract 9 Ovary 9 Cervix 8 Oesophagus 6 Liver 6 Upperaerodigestive tract 6 CNS 5 NS 5 Lung 4 Thyroid 4 Pituitary 3Soft_tissue 3 Pancreas 3 Kidney 2 Prostate 2 Meninges 1 Eye 1 Autonomicganglia 1 Haematopoietic/lymphoid 1 Adrenal_gland 0 Bone 0Fallopian_tube 0 Gastrointestinal_tract_(site_indeterminate) 0Peritoneum 0 Salivary_gland 0 Testis 0 Thymus 0 Vagina 0

As will be evident to anyone skilled in the art, this frequency data iscontinually being refined and updated as new and more comprehensive dataemerges from Human Cancer Genome profiling consortia such as the TCGA(The Cancer Genome Atlas) and ICGC (International Cancer GenomeConsortium). Hence additional tumour types with PIK3CA dependency may beidentified and be eligible for treatment with the compounds describedherein.

Methods for Detection of Nucleic Acids

The detection of mutant PIK3CA nucleic acids can be employed, in thecontext of the present invention, to predict the response to drugtreatment. Since mutations in these genes occur at the DNA level, themethods of the invention can be based on detection of mutations orvariances in genomic DNA, as well as transcripts and proteinsthemselves. It can be desirable to confirm mutations in genomic DNA byanalysis of transcripts and/or polypeptides, in order to ensure that thedetected mutation is indeed expressed in the subject.

It will be apparent to the person skilled in the art that there are alarge number of analytical procedures which may be used to detect thepresence or absence of variant nucleotides at one or more positions in agene. In general, the detection of allelic variation requires a mutationdiscrimination technique, optionally an amplification reaction (such asone based on polymerase chain reaction) and optionally a signalgeneration system. There are a multitude of mutation detectiontechniques available in the art and these may be used in combinationwith a signal generation system, of which there are numerous availablein the art. Many methods for the detection of allelic variation arereviewed by Nollau et al., Clin. Chem., 1997, 43, 1114-1120; Anderson SM. Expert Rev Mol Diagn., 2011, 11, 635-642; Meyerson M. et al., Nat RevGenet., 2010, 11, 685-696; and in standard textbooks, for example“Laboratory Protocols for Mutation Detection”, Ed. by U. Landegren,Oxford University Press, 1996 and “PCR”, 2^(nd) Edition by Newton &Graham, BIOS Scientific Publishers Limited, 1997.

As noted above, determining the presence or absence of a particularvariance or plurality of variances in the PIK3CA gene in a patient withcancer can be performed in a variety of ways. Such tests are commonlyperformed using DNA or RNA collected from biological samples, e.g.,tissue biopsies, urine, stool, sputum, blood, cells, tissue scrapings,breast aspirates or other cellular materials, and can be performed by avariety of methods including, but not limited to, PCR, hybridizationwith allele-specific probes, enzymatic mutation detection, chemicalcleavage of mismatches, mass spectrometry or DNA sequencing, includingminisequencing.

Suitable mutation detection techniques include amplification refractorymutation system (ARMS™), amplification refractory mutation system linearextension (ALEX™), competitive oligonucleotide priming system (COPS),Taqman, Molecular Beacons, restriction fragment length polymorphism(RFLP), and restriction site based PCR and fluorescence resonance energytransfer (FRET) techniques.

In particular embodiments the method employed for determining thenucleotide(s) within a biomarker gene is selected from: allele-specificamplification (allele specific PCR)—such as amplification refractorymutation system (ARMS), sequencing, allelic discrimination assay,hybridisation, restriction fragment length polymorphism (RFLP) oroligonucleotide ligation assay (OLA).

In particular embodiments, hybridization with allele specific probes canbe conducted by: (1) allele specific oligonucleotides bound to a solidphase (e.g. glass, silicon, nylon membranes) with the labelled sample insolution, for example as in many DNA chip applications; or, (2) boundsample (often cloned DNA or PCR amplified DNA) and labelledoligonucleotides in solution (either allele specific or short so as toallow sequencing by hybridization). Diagnostic tests may involve a panelof variances, often on a solid support, which enables the simultaneousdetermination of more than one variance. Such hybridization probes arewell known in the art (see, e.g., Green & Sambrook, Eds., MolecularCloning: A Laboratory Manual, (2012, 4th edition, Vol. 1-3, ISBN9781936113422), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) and may span two or more variance sites.

Thus, in one embodiment, the detection of the presence or absence of atleast one mutation provides for contacting PIK3CA nucleic acidcontaining a putative mutation site with at least one nucleic acidprobe. The probe preferentially hybridizes with a nucleic acid sequenceincluding a variance site and containing complementary nucleotide basesat the variance site under selective hybridization conditions.Hybridization can be detected with a detectable label using labels knownto one skilled in the art. Such labels include, but are not limited toradioactive, fluorescent, dye, and enzymatic labels.

In another embodiment, the detection of the presence or absence of atleast one mutation provides for contacting PIK3CA nucleic acidcontaining a putative mutation site with at least one nucleic acidprimer. The primer preferentially hybridizes with a nucleic acidsequence including a variance site and containing complementarynucleotide bases at the variance site under selective hybridizationconditions.

Oligonucleotides used as primers for specific amplification may carrythe complementary nucleotide base to the mutation of interest in thecentre of the molecule (so that amplification depends on differentialhybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res., 17,2437-248) or at the extreme 3′-terminus of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (see, e.g., Prossner, 1993, Tibtech, 11 238).

In yet another embodiment, the detection of the presence or absence ofat least one mutation comprises sequencing at least one nucleic acidsequence and comparing the obtained sequence with the known wild typenucleic acid sequence.

Alternatively, the presence or absence of at least one mutationcomprises mass spectrometric determination of at least one nucleic acidsequence.

In one embodiment, the detection of the presence or absence of at leastone nucleic acid variance comprises performing a polymerase chainreaction (PCR). The target nucleic acid sequence containing thehypothetical variance is amplified and the nucleotide sequence of theamplified nucleic acid is determined. Determining the nucleotidesequence of the amplified nucleic acid comprises sequencing at least onenucleic acid segment.

Alternatively, amplification products can be analyzed using any methodcapable of separating the amplification products according to theirsize, including automated and manual gel electrophoresis, and the like.

Mutations in genomic nucleic acid are advantageously detected bytechniques based on mobility shift in amplified nucleic acid fragments.For instance, Chen et al., Anal Biochem 1996, 239, 61-9, describe thedetection of single-base mutations by a competitive mobility shiftassay. Moreover, assays based on the technique of Marcelino et al.,BioTechniques 1999, 26, 1134-1148 are available commercially.

In a particular example, capillary heteroduplex analysis may be used todetect the presence of mutations based on mobility shift of duplexnucleic acids in capillary systems as a result of the presence ofmismatches.

Generation of nucleic acids for analysis from samples generally requiresnucleic acid amplification. Many amplification methods rely on anenzymatic chain reaction (such as a polymerase chain reaction, a ligasechain reaction, or a self-sustained sequence replication) or from thereplication of all or part of the vector into which it has been cloned.Preferably, the amplification according to the invention is anexponential amplification, as exhibited by for example the polymerasechain reaction.

Many target and signal amplification methods have been described in theliterature, for example, general reviews of these methods in Landegren,U., et al., Science, 1988 242, 229-237 and Lewis, R., GeneticEngineering News 1990, 10, 54-55. These amplification methods can beused in the methods of our invention, and include polymerase chainreaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligasehybridisation, QP3 bacteriophage replicase, transcription-basedamplification system (TAS), genomic amplification with transcriptsequencing (GAWTS), nucleic acid sequence-based amplification (NASBA)and in situ hybridisation. Primers suitable for use in variousamplification techniques can be prepared according to methods known inthe art.

Polymerase Chain Reaction (PCR) PCR is a nucleic acid amplificationmethod described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.PCR consists of repeated cycles of DNA polymerase generated primerextension reactions. The target DNA is heat denatured and twooligonucleotides, which bracket the target sequence on opposite strandsof the DNA to be amplified, are hybridised. These oligonucleotidesbecome primers for use with DNA polymerase. The DNA is copied by primerextension to make a second copy of both strands. By repeating the cycleof heat denaturation, primer hybridisation and extension, the target DNAcan be amplified a million fold or more in about two to four hours. PCRis a molecular biology tool, which must be used in conjunction with adetection technique to determine the results of amplification. Anadvantage of PCR is that it increases sensitivity by amplifying theamount of target DNA by 1 million to 1 billion fold in approximately 4hours. PCR can be used to amplify any known nucleic acid in a diagnosticcontext (Mok et al., Gynaecologic Oncology, 1994, 52: 247-252,).

An allele specific amplification technique such as AmplificationRefractory Mutation System (ARMS™) (Newton et al., Nucleic Acids Res.,1989, 17, 2503-2516) can also be used to detect single base mutations.Under the appropriate PCR amplification conditions a single basemismatch located at the 3′-end of the primer is sufficient forpreferential amplification of the perfectly matched allele (Newton etal., 1989, supra), allowing the discrimination of closely relatedspecies. The basis of an amplification system using the primersdescribed above is that oligonucleotides with a mismatched 3′-residuewill not function as primers in the PCR under appropriate conditions.This amplification system allows genotyping solely by inspection ofreaction mixtures after agarose gel electrophoresis.

Analysis of amplification products can be performed using any methodcapable of separating the amplification products according to theirsize, including automated and manual gel electrophoresis, massspectrometry, and the like.

The methods of nucleic acid isolation, amplification and analysis areroutine for one skilled in the art and examples of protocols can befound, for example, Green & Sambrook, Eds., Molecular Cloning: ALaboratory Manual, (2012, 4th edition, Vol. 1-3, ISBN 9781936113422),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)Particularly useful protocol source for methods used in PCRamplification is PCR (Basics: From Background to Bench) by M. J.McPherson, S. G. Mailer, R. Beynon, C. Howe, Springer Verlag; 1stedition (Oct. 15, 2000), ISBN: 0387916008.

The present invention also provides predictive and diagnostic kitscomprising degenerate primers to amplify a target nucleic acid in thePIK3CA gene and instructions comprising; amplification protocol andanalysis of the results. The kit may alternatively also comprisebuffers, enzymes, and containers for performing the amplification andanalysis of the amplification products. The kit may also be a componentof a screening, or diagnostic kit comprising other tools such as DNAmicroarrays, or other supports. Preferably, the kit also provides one ormore control templates, such as nucleic acids isolated from normaltissue sample, and/or a series of samples representing differentvariances in the reference genes.

In one embodiment, the kit provides two or more primer pairs, each paircapable of amplifying a different region of the reference (PIK3CA) gene(each region a site of potential variance) thereby providing a kit foranalysis of expression of several gene variances in a biological samplein one reaction or several parallel reactions.

Primers in the kits may be labelled, for example fluorescently labelled,to facilitate detection of the amplification products and consequentanalysis of the nucleic acid variances. The kit may also allow for morethan one variance to be detected in one analysis. A combination kit willtherefore comprise of primers capable of amplifying different segmentsof the reference gene. The primers may be differentially labelled, forexample using different fluorescent labels, so as to differentiatebetween the variances.

Also disclosed are use of ‘kits for the detection of PIK3CA mutations,including but not limited to, the PIK3CA mutation detection kitsmarketed by diagnostic companies including Qiagen and Roche MolecularSystems.

In another aspect, the invention provides a method of treating a patientsuffering from cancer comprising: determining the mutant or wild typestatus of the PIK3CA gene in the patient's tumour cells and if thePIK3CA gene is mutant, administering to the patient an effective amountof a compound of Formula (I).

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. “Lesseffective” means that the treatment results in a therapeuticallysignificant lower level of pharmacological effectiveness and/or atherapeutically greater level of adverse physiological effects.

According to another aspect of the invention there is provided the useof a compound of Formula (I) to treat a cancer patient whose tumourcells have been identified as possessing a mutant PIK3CA gene.

According to another aspect of the invention there is provided acompound of Formula (I) for treating cancers with tumour cellsidentified as harbouring mutant PIK3CA gene.

In still further embodiments, the invention relates to pharmaceuticalcomposition comprising a compound of Formula (I) for use in theprevention and treatment of cancer with tumour cells identified asharbouring a mutant PIK3CA gene.

For all the aspects above, mutant forms of PIK3CA determined/identifiedare at all positions across the gene.

For all the aspects above, using tumours such as breast cancer as anexample, particular mutant forms of PIK3CA determined/identified arethose at positions R38, R88, N345, C420, E453, P539, E542K, E545K, Q546,M1043, M1043 and H1047R,

For all the aspects above, using tumours such as breast cancer as anexample, particular mutant forms of PIK3CA determined/identified arethose at positions E542, E545 and H1047.

Personalised Healthcare/Personalised Medicine Examples CellProliferation Assay in Tumour Cell Lines

The sensitivity of a panel of human cancer cell lines to the effects ofcompounds was determined in a standard proliferation assay. Assayprotocol details are captured under Biological assays (g), above.

Mutation Correlation Analysis Methods

Pharmacology data measuring cell growth inhibition in response totreatment with Example 3 was obtained for a collection of 209 cancercell lines from a variety of tissues and from multiple sources. Eachcell line was classified as sensitive (GI50<=1.0 μM), or resistant(GI50>1.0 μM).

Mutation status for genes in each cell line was obtained by integratingresults from internal (AstraZeneca) and public sources. Public dataincluded all cell line data from the Genomics of Drug Sensitivity inCancer Project release 3 (Garnett M J, et al. Nature, 2012, March 483,570-5), Cancer Cell Line Encyclopedia project (Barretina J, et al.,Nature 2012, 483, 603-7) and the Catalogue of Somatic Mutations InCancer (COSMIC) database (release v61;http://www.sanger.ac.uk/genetics/CGP/cosmic/; Forbes S A, et al. NucleicAcids Res, 2011, 39 (Database issue):D945-50; Forbes S A, et al., CurrProtoc Hum Genet. 2008; Chapter 10:Unit 10.11.), and selected journalarticles. Silent coding region mutations (synonymous variants) andnon-synonymous polymorphisms were excluded, and, for the purpose of thisanalysis, the zygosity of mutations was ignored. For each combination ofcell line and gene, status was summarized as mutant (MUT), wild-type(WT) or inconsistent (INCON). Some initially inconsistent cases(independent WT and MUT observations for the same gene in the same cellline) were resolved by weighting internal observations and those for theCancer Cell line Project (CCLP) subset of COSMIC, or by selecting astatus after manual review. In cases where inconsistent observationscould not be resolved, the INCON label was retained and the gene statuswas regarded as unknown during analysis.

Associations between mutation status and response were identified byconstructing contingency tables for each gene and determiningcorresponding odd-ratios and two-tailed Fisher's exact test p-values.The cell lines classified as marginal for response were excluded fromthe initial analyses to identify candidate biomarkers. For mutationstatus, MUT or WT findings were counted and Genes with fewer than 4 WTor 4 MUT cell lines were also excluded.

Results and Discussion

Associations between mutation status and response were identified asdescribed in Methods. The cell line response to Example 3 and thecorresponding genetic status for the PIK3CA gene is shown in Table 3.

TABLE 3 The pharmacology data, response classification and the mutationstatus of the PIK3CA gene for the cell lines used in this study. CellLine Category Tissue GI50 μM PIK3CA RS411 Sensitive Blood/lymph 9.03E−02WT T47D Sensitive Breast 0.1982 MUT H596 Sensitive Lung 0.3018 MUT MCF7Sensitive Breast 0.3094 MUT MV411 Sensitive Blood/lymph 0.3816 WT HRA19Sensitive Rectum 0.3926 MUT IM95M Sensitive Stomach 0.4359 MUT MDAMB453Sensitive Breast 0.4564 MUT JEKO1 Sensitive Blood/lymph 0.4994 WT SNU601Sensitive Stomach 0.5063 MUT HCC1187 Sensitive Breast 0.5088 WT SW48Sensitive Colon 0.5131 MUT H1703 Sensitive Lung 0.5144 WT THP1 SensitiveBlood/lymph 0.5281 WT LK2 Sensitive Lung 0.5884 WT HUPT4 SensitivePancreas 0.6408 WT L363 Sensitive Blood/lymph 0.6812 MUT TCCSUPSensitive Bladder 0.7237 MUT VMCUB1 Sensitive Bladder 0.7319 MUTRERFLCSQ1 Sensitive Lung 0.7711 MUT HCC1419 Sensitive Breast 0.7799 WTLNCAPCASRES Sensitive Prostate 0.7924 WT CCK81 Sensitive Colon 0.8031MUT HCC1954 Sensitive Breast 0.8794 MUT SW948 Sensitive Colon 0.9111 MUTPANC0203 Sensitive Pancreas 0.9628 WT BFTC905 Sensitive Bladder 0.9662WT REH Resistant Blood/lymph 1.047 WT SNU216 Resistant Stomach 1.072 WTSKCO1 Resistant Colon 1.128 WT SUM52PE Resistant Breast 1.145 WT RT11284Resistant Bladder 1.171 WT OVCAR3 Resistant Ovary 1.179 WT MOLM13Resistant Blood/lymph 1.22 WT C99 Resistant Colon 1.224 WT CALU3Resistant Lung 1.296 WT N87 Resistant Stomach 1.301 WT 2313287 ResistantStomach 1.339 WT PAMC82 Resistant Stomach 1.366 WT HCC1569 ResistantBreast 1.369 WT AGS Resistant Stomach 1.414 MUT JIMT1 Resistant Breast1.46 MUT HGC27 Resistant Stomach 1.501 MUT MKN1 Resistant Stomach 1.579MUT SKBR3 Resistant Breast 1.588 WT SNU368 Resistant Liver 1.597 WTPANC89 Resistant Pancreas 1.618 WT ASPC1 Resistant Pancreas 1.74 WTSNU484 Resistant Stomach 1.785 WT H2085 Resistant Lung 1.835 WT HARAResistant Lung 1.906 WT AZ521 Resistant Duodenum 2.063 WT HPAC ResistantPancreas 2.162 WT NOMO1 Resistant Blood/lymph 2.167 WT PNT1A ResistantProstate 2.17 WT H1975 Resistant Lung 2.262 MUT OCUM1 Resistant Stomach2.332 WT BT20 Resistant Breast 2.36 MUT HCT8 Resistant Colon 2.51 MUTCOLO320DM Resistant Colon 2.512 WT PANC1005 Resistant Pancreas 2.607 WTSW403 Resistant Colon 2.61 MUT MONOMAC6 Resistant Blood/lymph 2.622 WTHPAFII Resistant Pancreas 2.63 WT HT1197 Resistant Bladder 2.8 MUTLNCAPCLONEFGC Resistant Prostate 3.007 WT HCC95 Resistant Lung 3.107 WTSNU620 Resistant Stomach 3.144 WT MOLP8 Resistant Blood/lymph 3.289 WTH2291 Resistant Lung 3.291 WT DMS114 Resistant Lung 3.294 WT MHCC97LResistant Liver 3.353 WT CFPAC1 Resistant Pancreas 3.384 WT HS766TResistant Pancreas 3.467 WT ZR751 Resistant Breast 3.558 WT PC3Resistant Prostate 3.833 WT 22RV1 Resistant Prostate 4.144 MUT RKOResistant Colon 4.323 MUT 977 Resistant Bladder 4.42 WT MOLM16 ResistantBlood/lymph 4.601 WT H358 Resistant Lung 4.642 WT LUDLU1 Resistant Lung4.646 WT QGP1 Resistant Pancreas 4.865 WT OE19 Resistant Esophagus 5.129WT SW1710 Resistant Bladder 5.339 WT PANC1 Resistant Pancreas 5.344 WTSNU449 Resistant Liver 5.41 WT 647V Resistant Bladder 5.464 WT HT29Resistant Colon 5.483 MUT SNU354 Resistant Liver 5.604 WT HS746TResistant Stomach 5.978 WT H1869 Resistant Lung 6.044 WT UMUC3 ResistantBladder 6.217 WT PANC0403 Resistant Pancreas 6.468 WT KG1 ResistantBlood/lymph 6.588 WT H520 Resistant Lung 6.619 WT HEP3B Resistant Liver6.687 WT HCT15 Resistant Colon 7.268 MUT H1793 Resistant Lung 7.329 WTU937 Resistant Blood/lymph 7.345 WT H2170 Resistant Lung 7.644 WTPANC0327 Resistant Pancreas 8.025 WT BEL7405 Resistant Liver 8.11 WTHT1376 Resistant Bladder 8.199 WT SNU638 Resistant Stomach 8.221 WT H322Resistant Lung 8.227 WT DU145 Resistant Prostate 8.239 WT EBC1 ResistantLung 8.566 WT JURKAT Resistant Blood/lymph 8.691 WT COLO205 ResistantColon 8.934 WT RT4 Resistant Bladder 8.936 WT KATOIII Resistant Stomach9.155 WT MDAMB468 Resistant Breast 9.325 WT 5637 Resistant Bladder 9.627WT OE33 Resistant Esophagus 9.856 WT LS180 Resistant Colon 9.942 MUTHCCC9810 Resistant Bile duct 10.02 WT H226 Resistant Lung 10.1 WT A549Resistant Lung 10.15 WT QGY7703 Resistant Liver 11.07 WT H647 ResistantLung 11.34 WT MGHU3 Resistant Bladder 11.5 WT H23 Resistant Lung 12.3 WTSCABER Resistant Bladder 12.4 WT H2126 Resistant Lung 12.91 WT HUPT3Resistant Pancreas 13.39 WT SW620 Resistant Colon 13.4 WT CAPAN2Resistant Pancreas 13.42 WT J82 Resistant Bladder 13.42 MUT HLEResistant Liver 13.47 WT BXPC3 Resistant Pancreas 14.08 WT MCF7MDR+Resistant Breast 14.45 WT BEL7404 Resistant Liver 14.9 WT SNU1 ResistantStomach 14.97 WT KP4 Resistant Pancreas 15.05 WT CAMA1 Resistant Breast15.47 WT HCA7 Resistant Colon 15.49 WT SNU668 Resistant Stomach 15.51 WTH522 Resistant Lung 15.55 WT SNU886 Resistant Liver 15.6 WT SW480Resistant Colon 15.9 WT HUH7 Resistant Liver 15.97 WT CALU1 ResistantLung 16.03 WT SNU878 Resistant Liver 16.06 WT HCC1806 Resistant Breast16.71 WT SNU16 Resistant Stomach 16.76 WT GTL16 Resistant Stomach 17.38WT BT549 Resistant Breast 17.44 WT NAMALWA Resistant Blood/lymph 17.55WT WSUDLCL2 Resistant Blood/lymph 17.71 WT SU8686 Resistant Pancreas17.97 WT H460DNP53 Resistant Lung 17.98 WT SNU761 Resistant Liver 18.49WT LOVO Resistant Colon 18.64 WT SW780 Resistant Bladder 19.23 WT SKMES1Resistant Lung 19.54 WT H2286 Resistant Lung 20.03 WT SNU5 ResistantStomach 21.19 WT HCC1395 Resistant Breast 21.81 WT HUH1 Resistant Liver22.34 WT MDAMB231 Resistant Breast 23.61 WT NUGC3 Resistant Stomach24.15 WT MIAPACA2 Resistant Pancreas 24.2 WT SNU739 Resistant Liver25.91 WT CALU6 Resistant Lung 26.15 WT AMO1 Resistant Blood/lymph 26.93WT SW1990 Resistant Pancreas 28.28 WT CMK Resistant Blood/lymph 28.91 WT1A6 Resistant Bladder 30 WT A2058 Resistant Skin 30 WT ARH77 ResistantBlood/lymph 30 WT CAPAN1 Resistant Pancreas 30 WT CC20 Resistant Colon30 WT H1299 Resistant Lung 30 WT H1437 Resistant Lung 30 WT H460Resistant Lung 30 MUT H526 Resistant Lung 30 WT H838 Resistant Lung 30WT HCC15 Resistant Lung 30 WT HCC1937 Resistant Breast 30 WT HCT116Resistant Colon 30 MUT HEL9217 Resistant Blood/lymph 30 INCON HEPG2Resistant Liver 30 WT HLF Resistant Liver 30 WT HX147 Resistant Lung 30WT IM9 Resistant Blood/lymph 30 WT JJN3 Resistant Blood/lymph 30 WT JVM3Resistant Blood/lymph 30 WT K562 Resistant Blood/lymph 30 WT KU1919Resistant Bladder 30 WT MDAMB157 Resistant Breast 30 WT MDAMB436Resistant Breast 30 WT MEC1 Resistant Blood/lymph 30 WT MKN74 ResistantStomach 30 WT NUGC4 Resistant Stomach 30 WT OCIAML2 ResistantBlood/lymph 30 WT OCILY19 Resistant Blood/lymph 30 WT PC9 Resistant Lung30 WT RAJI Resistant Blood/lymph 30 WT RAMOS Resistant Blood/lymph 30 WTRERFLCAI Resistant Lung 30 WT RPMI8226 Resistant Blood/lymph 30 WT SC1Resistant Blood/lymph 30 WT SKHEP1 Resistant Liver 30 WT SMMC7721Resistant Liver 30 WT SNU398 Resistant Liver 30 WT SW900 Resistant Lung30 WT T24 Resistant Bladder 30 WT YAPC Resistant Pancreas 30 WT

The gene for which mutations were most strongly correlated withsensitivity to Example 3 was PIK3CA. Only 12 of 177 PIK3CA WT cell lines(7.7%) were sensitive to Example 3, whereas 15 of 32 cell lines (46.9%)that are mutant for PIK3CA were sensitive, corresponding to anodds-ratio of 12.1 and a p-value of 1.2×10⁻⁷ (see Table 4).

TABLE 4 Contingency table for PIK3CA mutation status and response toExample 3. Mutation Status Response (PIK3CA) Sensitive Resistant MUT 1517 WT 12 165 Odds-Ratio: 12.1 p-value: 1.2 × 10⁻⁷

As indicated herein, it has been reported that the measurement ofmutation status or activation status of additional genes, such as KRAS,a potential marker of resistance in tumours with aberrant or deregulatedPIK3CA or PI3K-ε, could help increase the predictivity of a PersonalisedMedicine approach.

We exemplified this for the above dataset by comparing the enrichment ofKRAS mutations in PIK3CA mutant cells with the cell line's response toinhibition. Analysis was limited to cell lines containing ‘hotspot’mutations of the two genes (at codons E542, E545 and H1047 for PIK3CAand at codons K12, 13 and Q61 for KRAS). This demonstrated that inPIK3CA mutant cell lines, mutations in KRAS conferred resistance toinhibition by Example 3.

-   -   Twenty-eight cell lines contained activating mutations in        PIK3CA.    -   6 of 19 cell lines (31.6%) containing an activating PIK3CA        mutation and a wild-type KRAS gene were resistant to Example 3.    -   7 of 9 PIK3CA mutant cell lines (77.8%) contained coexisting        KRAS mutations and were resistant to Example 3.        This translates into an odds-ratio of 7.5 and a p-value of 0.042        (see Table 5).

TABLE 5 Contingency table for PIK3CA and KRAS mutation status andresponse to Example 3. Mutation Response Status Sensitive Resistant KRASAND 2 7 PIK3CA MUT PIK3CA MUT 13 6 and KRAS WT Odds Ratio: 7.5 p-value:0.042

EXAMPLES

The invention will now be illustrated in the following Examples inwhich, generally:

(i) operations were carried out at ambient temperature, i.e. in therange 17 to 25° C. and under an atmosphere of an inert gas such asnitrogen unless otherwise stated;

(ii) evaporations were carried out by rotary evaporation or utilisingGenevac equipment in vacuo and work-up procedures were carried out afterremoval of residual solids by filtration;

(iii) Flash chromatography purifications were performed on an automatedArmen Glider Flash: Spot II Ultimate (Armen Instrument, Saint-Ave,France) using prepacked Merck normal phase Si60 silica cartridges(granulometry: 15-40 or 40-63 μm) obtained from Merck, Darmstad,Germany;

(iv) preparative chromatography was performed on a Waters instrument(600/2700 or 2525) fitted with a ZMD or ZQ ESCi mass spectrometers and aWaters X-Terra or a Waters X-Bridge or a Waters SunFire reverse-phasecolumn (C-18, 5 microns silica, 19 mm diameter, 100 mm length, flow rateof 40 mL/minute) using decreasingly polar mixtures of water (containing0.2% ammonium carbonate) and acetonitrile as eluent;

(v) yields, where present, are not necessarily the maximum attainable;

(vi) in general, the structures of end-products of the Formula I wereconfirmed by nuclear magnetic resonance (NMR) spectroscopy; NMR chemicalshift values were measured on the delta scale [proton magnetic resonancespectra were determined using a Bruker Avance 500 (500 MHz) instrument];measurements were taken at ambient temperature unless otherwisespecified; the following abbreviations have been used: s, singlet; d,doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets;ddd, doublet of doublet of doublet; dt, doublet of triplets; bs, broadsignal;

(vii) in general, end-products of the Formula I were also characterisedby mass spectroscopy following liquid chromatography (LCMS); LCMS wascarried out using an Waters Alliance HT (2790 & 2795) fitted with aWaters ZQ ESCi or ZMD ESCi mass spectrometer and an X Bridge 5 μm C-18column (2.1×50 mm) at a flow rate of 2.4 mL/min, using a solvent systemof 95% A+5% C to 95% B+5% C over 4 minutes, where A=water, B=methanol,C=1:1 methanol:water (containing 0.2% ammonium carbonate);

(viii) intermediates were not generally fully characterised and puritywas assessed by thin layer chromatographic, mass spectral, HPLC and/orNMR analysis;

(ix) X-ray powder diffraction spectra were determined (using a Bruker D4Analytical Instrument) by mounting a sample of the crystalline materialon a Bruker single silicon crystal (SSC) wafer mount and spreading outthe sample into a thin layer with the aid of a microscope slide. Thesample was spun at 30 revolutions per minute (to improve countingstatistics) and irradiated with X-rays generated by a copper long-finefocus tube operated at 40 kV and 40 mA with a wavelength of 1.5418angstroms. The collimated X-ray source was passed through an automaticvariable divergence slit set at V20 and the reflected radiation directedthrough a 5.89 mm antiscatter slit and a 9.55 mm detector slit. Thesample was exposed for 0.03 seconds per 0.00570° 2-theta increment(continuous scan mode) over the range 2 degrees to 40 degrees 2-theta intheta-theta mode. The running time was 3 minutes and 36 seconds. Theinstrument was equipped with a Position sensitive detector (Lynxeye).Control and data capture was by means of a Dell Optiplex 686 NT 4.0Workstation operating with Diffrac+software. Persons skilled in the artof X-ray powder diffraction will realise that the relative intensity ofpeaks can be affected by, for example, grains above 30 microns in sizeand non-unitary aspect ratios that may affect analysis of samples. Theskilled person will also realise that the position of reflections can beaffected by the precise height at which the sample sits in thediffractometer and the zero calibration of the diffractometer. Thesurface planarity of the sample may also have a small effect. Hence thediffraction pattern data presented are not to be taken as absolutevalues;

(x) Differential Scanning Calorimetry was performed using a TAInstruments Q1000 DSC instrument. Typically less than 5 mg of materialcontained in a standard aluminium pan fitted with a lid was heated overthe temperature range 25° C. to 300° C. at a constant heating rate of10° C. per minute. A purge gas using nitrogen was used at a flow rate of50 mL per minute; and

(xi) the following abbreviations have been used:—

-   -   aq. aqueous    -   CDCl₃ deutero-chloroform    -   CHCl₃ chloroform    -   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCM dichloromethane    -   DEA diethyl amine    -   DIPEA N-ethyl-N-isopropylpropan-2-amine    -   DMA N,N-dimethylacetamide    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulphoxide    -   DSC Differential Scanning Calorimetry    -   DTAD (E)-di-tert-butyl diazene-1,2-dicarboxylate    -   EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   Ether diethyl ether    -   EtOH ethanol    -   EtOAc ethyl acetate    -   % ee % enantiomeric excess    -   HOPO 2-hydroxy-pyridine N-oxide    -   HPLC high performance liquid chromatography    -   IPA isopropyl alcohol    -   MeCN acetonitrile    -   MeOH methanol    -   MIBK methyl isobutyl ketone    -   MTBE methyl tert-butyl ether    -   NMP 1-methyl-2-pyrrolidone    -   rt room temperature    -   sat. saturated    -   sol. solution    -   THF tetrahydrofuran    -   TEA triethyl amine    -   TBTU        2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium        tetrafluoroborate    -   v/v volume/volume    -   TFA trifluoroacetic acid

Example 11-(4-(5-(5-Amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

3-Hydroxypropanoic acid (30% v/v soln in water) (200 μL, 47.0 mg, 0.52mmol) was evaporated to dryness then azeotroped with the toluene. Theacid was dissolved in NMP (1 mL) and molecular sieves (100 mg, 0.26mmol), N-ethyl-N-isopropylpropan-2-amine (0.136 mL, 0.78 mmol) wereadded followed by the addition of2-(1H-benzo[d][1,2,3]-triazol-1-yl)-1,1,3,3-tetramethylisouroniumtetrafluoroborate (209 mg, 0.65 mmol). After 30 minutes stirring,3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(100 mg, 0.26 mmol) was added and the mixture was stirred for 2 hours.The reaction mixture was purified by preparative HPLC using a WatersX-Bridge reverse-phase column (C-18, 5 microns silica, 19 mm diameter,100 mm length, flow rate of 40 ml/minute) and decreasingly polarmixtures of water (containing 0.2% ammonium carbonate) and acetonitrileas eluent.

The fractions containing the desired compound were evaporated to drynessto afford1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(45.0 mg, 37.9%) as a clear yellow solid: ¹H NMR Spectrum (CDCl3) 1.52(9H, s), 1.79-1.94 (2H, m), 2.07-2.15 (2H, m), 2.58 (2H, t), 2.84-2.94(1H, m,), 3.00-3.10 (1H, m), 3.17-3.26 (1H, m), 3.53 (1H, t), 3.86-3.94(3H, m), 4.30 (3H, s), 4.56-4.62 (1H, m), 9.02 (1H, s); Mass Spectrum[M+H]⁺=456.

3-(5-tert-Butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(Example 1.1) was prepared as follows:

At 20° C., tert-butyl 4-carbamoylpiperidine-1-carboxylate (47 g, 205.88mmol) in dichloromethane (500 mL) was added dropwise to a stirredsolution of triethyloxonium hexafluorophosphate(V) (56.2 g, 226.47 mmol)in dichloromethane (500 mL) over a period of 45 minutes under nitrogen.The resulting solution was stirred at 20 OC overnight. A saturatedaqueous solution of Na₂CO₃ was then added until pH of 8 was obtained.The phases were decanted and the aqueous phase was extracted again with200 mL of CH₂Cl₂ then the organic phases were dried over MgSO₄, filteredand concentrated to afford tert-butyl4-(ethoxy(imino)methyl)piperidine-1-carboxylate (51.0 g, 97%) as acolourless liquid: ¹H NMR Spectrum; (CDCl₃) 1.28 (3H, t), 1.46 (9H, s),1.47 (2H, m), 1.79-1.93 (2H, m), 2.28 (1H, m), 2.73 (2H, m), 4.10 (2H,q), 4.13-4.18 (2H, m); Mass Spectrum [M+H]⁺=no mass ion.

To a stirred solution of tert-butyl4-(ethoxy(imino)methyl)piperidine-1-carboxylate (51 g, 198.95 mmol) indioxane (500 mL), was added formohydrazide (17.92 g, 298.43 mmol). Thissolution was left to stir at 40° C. overnight under N₂ resulting inprecipitation of a white solid (hydrazide intermediate). The reactionmixture was then heated to 80° C. for 6 h, cooled to room temperatureand concentrated. The residue was dissolved in 500 mL of CH₂Cl₂ and 300mL of water was added. The phases were decanted and the organic phasewas washed with brine, dried over MgSO₄, filtered and concentrated toafford tert-butyl 4-(1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(46.0 g, 92%) as a white solid: ¹H NMR Spectrum; (CDCl₃) 1.47 (9H, s),1.76 (2H, m), 1.98-2.11 (2H, m), 2.91 (2H, s), 2.97-3.08 (m, 1H),4.06-4.23 (2H, m), 8.05 (1H, s); Mass Spectrum [M+H]⁺=no mass ion.

To a stirred solution of tert-butyl4-(1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (22 g, 87.19 mmol) indichloromethane (250 mL) was added sodium hydroxide 2N (131 mL, 261.58mmol). The reaction mixture was vigorously stirred with mechanicalstirring and a solution of benzyltrimethylammonium tribromide (37.4 g,95.91 mmol) in dichloromethane (250 mL) was then added dropwise, keepingtemperature around 15° C.

The reaction mixture was left to stir at room temperature for 1 h and 2NHCl was added to give a pH of 5 (keeping temperature around 15° C.). Thephases were decanted and the organic phase was washed with H₂O (2×1 L),dried over MgSO₄, filtered and concentrated to afford tert-butyl4-(5-bromo-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (25.00 g, 87%)as an off-white solid: ¹H NMR Spectrum; (CDCl₃) 1.46 (9H, s), 1.67-1.84(2H, m), 1.90-2.13 (2H, m), 2.77-2.96 (2H, m), 2.98-3.10 (1H, m),3.94-4.35 (2H, m); Mass Spectrum [M+H]⁺=no mass ion.

To a stirred suspension of tert-butyl4-(5-bromo-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (26 g, 78.50mmol) in toluene (200 mL) and methanol (50 mL) was added dropwise(diazomethyl)trimethylsilane 2M solution in hexane (43.2 mL, 86.35 mmol)under N₂, keeping temperature around 20° C.: gas evolution and a smallexotherm were observed. The yellow solution obtained was stirred at roomtemperature for 1 h. The solvent were evaporated and the resulting oilwas purified on silica, eluting with 40% EtOAc in petroleum ether toafford tert-butyl4-(5-bromo-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(15.00 g, 55.3%) as an oil: ¹H NMR Spectrum; (CDCl₃) 1.46 (9H, s),1.65-1.78 (2H, m), 1.90-2.01 (2H, m), 2.68-3.02 (3H, m), 3.83 (3H, s),3.94-4.31 (2H, m); Mass Spectrum [M+H]⁺=no mass ion.

Hydrazine monohydrate (34 mL, 1094.95 mmol) was added portionwise to astirred suspension of methyl 3-aminopyrazine-2-carboxylate (21.3 g,139.09 mmol) in ethanol (65 mL) at r.t. The resulting slurry was stirredat 60 OC for 2 hours, cooled to room temperature and filtered. The solidwas washed with cold ethanol (2×25 ml) and dried to a constant weight toafford 3-aminopyrazine-2-carbohydrazide (20.75 g, 97%) as a beige solid:¹H NMR Spectrum; (DMSO-d₆) 4.49 (2H, d), 7.46 (2H, br s,), 7.78 (1H, d),8.17 (1H, d), 9.79 (1H, t); Mass Spectrum [M+H]⁺=154.

2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouroniumtetrafluoroborate (47.7 g, 148.69 mmol) was added portionwise over 15minutes to a stirred suspension of N-ethyl-N-isopropylpropan-2-amine(70.6 mL, 405.51 mmol), pivalic acid (17.08 mL, 148.69 mmol) and3-aminopyrazine-2-carbohydrazide (20.7 g, 135.17 mmol) in acetonitrile(350 mL) and the reaction mixture was stirred at 80° C. for 20 minutes(a solution was obtained). The reaction mixture was cooled to 0 OC andN-ethyl-N-isopropylpropan-2-amine (70.6 mL, 405.51 mmol), followed by4-methylbenzene-1-sulfonyl chloride (77 g, 405.51 mmol) were added overa period of 15 minutes. The reaction mixture (yellow suspension) wasbrought to reflux (solubilisation) and then allowed to stir at roomtemperature for 14 hours affording a dark orange solution. The solutionwas concentrated. The residue was diluted with dichloromethane, washedwith water, brine, dried over magnesium sulfate and concentrated. Thecrude product was purified by flash chromatography on silica gel elutingwith 0 to 40% ethyl acetate in dichloromethane. The solvent wasevaporated to dryness. The resulting mixture was triturated with ether(100 mL), filtered, washed with the minimum of ether and dried to afford3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (20.8 g, 70.2%) asa pale yellow soild: ¹H NMR Spectrum; (CDCl₃) 1.53 (9H, s), 1.58-1.68(2H, m), 6.67 (2H, s), 8.13 (2H, dt); Mass Spectrum [M+H]⁺=220.

1-Bromopyrrolidine-2,5-dione (18.57 g, 104.36 mmol) was addedportionwise to a solution of3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (20.8 g, 94.87mmol) in THF (320 mL) and the solution was stirred at room temperaturefor 16 hours. The reaction mixture was concentrated and the residue wasdissolved in dichloromethane (300 mL), washed with water (2×150 mL),brine, dried over magnesium sulfate and concentrated. The solvent wasevaporated and the crude product was purified by flash chromatography onsilica gel, eluting with 0 to 10% ethyl acetate in dichloromethane. Thesolvent was evaporated to dryness to afford5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (25.5 g,90%) as a beige solid: ¹H NMR Spectrum; (CDCl₃) 1.52 (9H, s), 8.23 (1H,s); Mass Spectrum [M+H]⁺=300.

To a suspension of5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (45 g,150.94 mmol) in toluene (450 mL) were added1,1,1,2,2,2-hexamethyldistannane (37.6 mL, 181.12 mmol) andbis(triphenylphosphine)palladium (II) chloride (5.30 g, 7.55 mmol). Thereaction mixture was degassed with argon and heated at 80° C. for 2hours. (solubilisation upon heating, orange solution thenreprecipitation and turned black indicating reaction is complete.) Thereaction mixture was cooled down, concentrated and the residue wasdissolved in CH₂Cl₂ and filtered on Decalite to remove the insolubleimpurities. The filtrate was concentrated and purified on silica elutingwith 0 to 10% EtOAc in CH₂Cl₂. The solvent was concentrated to afford3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(trimethylstannyl)pyrazin-2-amine(22.63 g, 39.2%) as an orange solid: ¹H NMR Spectrum; (CDCl₃) 0.38 (9H,s), 1.53 (9H, s), 6.49 (2H, br s), 8.13 (1H, s); Mass Spectrum[M+H]⁺=384.

To a stirred suspension of tert-butyl4-(5-bromo-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (2700mg, 7.82 mmol) and3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(trimethylstannyl)pyrazin-2-amine(2988 mg, 7.82 mmol) in 4-methyl-2-pentanol (28 mL) were added lithiumchloride (995 mg, 23.46 mmol) and bis(triphenylphosphine)palladium (II)chloride (220 mg, 0.31 mmol). The mixture was degassed with argon andheated at 140° C. for 2 h. The reaction was cooled down and theresulting precipitate was collected by filtration, washed withisopropanol (25 mL), water (25 mL) and dried under suction. Theisopropanol organic fraction was concentrated and the precipitate formedwas collected and combined with the main precipitate affordingtert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(3.0 g, 79%): ¹H NMR Spectrum: (DMSO-d₆) 1.41 (9H, s), 1.45 (9H, s),1.50-1.68 (2H, m), 1.95 (3H, dd), 2.78-3.05 (1H, m), 3.96 (3H, d), 4.21(3H, s), 8.86 (1H, s); Mass Spectrum [M+H]⁺=484.

A solution of tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(3 g, 6.20 mmol) in TFA (15 mL) and CH₂Cl₂ (15 mL) was stirred at 25° C.for 1 hour. The mixture was azeotroped with toluene, a 7N solution ofammonia in methanol and dichloromethane were added and the mixture wasadsorbed on silica gel. The crude product was purified by flashchromatography on silica gel eluting with 0 to 8% methanol indichloromethane followed by 0 to 10% methanolic ammonia indichloromethane The solvent was evaporated to dryness to afford3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(2.040 g, 86%) as a yellow crystalline solid: ¹H NMR Spectrum: (DMSO-d₆)1.45 (9H, s), 1.55-1.66 (2H, m), 1.86 (2H, dd), 2.52-2.61 (2H, m),2.69-2.78 (1H, m), 2.95-3.02 (2H, m), 4.20 (3H, s), 8.86 (1H, s); MassSpectrum [M+H]⁺=384.

Isolation of single crystalline form of Example 1

The X-ray powder diffraction spectra of the material isolated aboveshowed the material to be crystalline but a mixture of polymorphicforms. This material had a melting point of 226.4° C. (onset).

Form A material was produced by slurrying the original material inacetonitrile at 25° C. Approximately 20 mg of the original material wasplaced in a vial with a magnetic stirrer bar, and approximately 2 mL ofacetonitrile added, the vial was then sealed tightly with a cap and leftto stir on a magnetic stirrer plate. After approximately 5 days, thesample was removed from the plate, the cap taken off and the slurry leftto dry under ambient conditions before it was analysed by XRPD and DSC.This form (Form A) was determined to be crystalline by XRPD. Thismaterial had a melting point of 227.2° C. (onset).

The same crystalline form may be made by stirring the crude material inacetonitrile overnight at room temperature, then filtering the resultingsolid, washing with cold acetonitrile and drying.

In one aspect of the invention there is provided a process for forming acrystalline form of Example 1 (Form A) by slurrying a sample of thecompound in acetonitrile.

Ten X-Ray powder diffraction peaks are shown in the Table below:

Ten X-Ray Powder Diffraction peaks for Example 1 Form A Angle 2- Theta(2θ) Intensity % 5.1 100.0 18.0 22.5 10.2 22.0 11.7 17.8 19.4 14.5 18.514.2 14.8 12.6 26.7 11.0 26.6 10.6 17.8 9.9

An X-Ray powder diffraction spectrum of Example 1 Form A is shown inFIG. 1.

DSC analysis of Example 1 Form A shows a melting endotherm with an onsetof 227.2° C. and a peak at 228.6° C. (FIG. 2).

Thus DSC analysis shows Example 1 Form A is a high melting solid with anonset of melting at about 227.2° C. and a peak at 228.6° C.

A DSC of Example 1 Form A is shown in FIG. 2.

X-Ray Powder Diffraction

Analytical Instrument: Bruker D4.

The X-ray powder diffractogram was determined by mounting a sample ofthe crystalline material on a Bruker single silicon crystal (SSC) wafermount and spreading out the sample into a thin layer with the aid of amicroscope slide. The sample was spun at 30 revolutions per minute (toimprove counting statistics) and irradiated with X-rays generated by acopper long-fine focus tube operated at 40 kV and 40 mA with awavelength of 1.5418 angstroms. The collimated X-ray source was passedthrough an automatic variable divergence slit set at V20 and thereflected radiation directed through a 5.89 mm antiscatter slit and a9.55 mm detector slit. The sample was exposed for 0.03 seconds per0.00570° 2-theta increment (continuous scan mode) over the range 2degrees to 40 degrees 2-theta in theta-theta mode. The running time was3 minutes and 36 seconds. The instrument was equipped with a Positionsensitive detector (Lynxeye). Control and data capture was by means of aDell Optiplex 686 NT 4.0 Workstation operating with Diffrac+software.Persons skilled in the art of X-ray powder diffraction will realise thatthe relative intensity of peaks can be affected by, for example, grainsabove 30 microns in size and non-unitary aspect ratios that may affectanalysis of samples. The skilled person will also realise that theposition of reflections can be affected by the precise height at whichthe sample sits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values.

Differential Scanning Calorimetry

Analytical Instrument: TA Instruments Q1000 DSC.

Typically less than 5 mg of material contained in a standard aluminiumpan fitted with a lid was heated over the temperature range 25° C. to300° C. at a constant heating rate of 10° C. per minute. A purge gasusing nitrogen was used—flow rate 50 ml per minute.

An alternative synthesis of the compound of Example 1 is provided belowas Example 2.

Example 21-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

Pyridine 4-methylbenzenesulfonate (3.58 g, 14.25 mmol) was added to asuspension of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(37 g, 68.57 mmol) in methanol (275 mL) under nitrogen. The mixture wasstirred at 60° C. for 1.5 hours. The mixture was soluble after 5minutes. The mixture was held at 50° C. overnight during which time aprecipitate formed. The reaction mixture was dissolved indichloromethane (400 mL), washed with water (300 mL), and brine (100mL). The aqueous extracts were backwashed with DCM (100 mL) and thecombined organic layers were dried over MgSO₄ and concentrated. Thecrude product was purified by flash chromatography on silica gel elutingwith 100% ethyl acetate to 10:50:40 methanol/ethyl acetate/DCM. Theproduct containing fractions were evaporated to dryness to afford abeige solid (24.5 g). The solid was slurried overnight in acetonitrile(500 mL), filtered and dried under high vacuum to afford1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(Example 2) (24 g, 78%) as a cream solid: ¹H NMR Spectrum: (DMSO-d₆)1.51 (9H, s), 1.55-1.68 (1H, m), 1.68-1.84 (1H, m), 1.96-2.13 (2H, m),2.78-2.93 (1H, m), 2.98-3.1 (1H, m), 3.19-3.3 (1H, m), 3.71 (2H, q),3.93-4.04 (1H, m), 4.27 (3H, s), 4.35-4.48 (1H, m), 4.54 (1H, t), 7.96(2H, s), 8.92 (1H, s); Mass Spectrum [M+H]⁺=456

(4-(5-(5-Amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(Example 2.1) was prepared as follows:

Hydrazine hydrate (23.59 mL, 480.75 mmol) was added dropwise to astirred mixture of methyl 3-amino-6-bromopyrazine-2-carboxylate (100 g,418.04 mmol) in EtOH (2 L). The mixture was heated at 50° C. undernitrogen. The resulting thick suspension was stirred at 50° C. for 16hours. Further hydrazine (2.5 mL) was added in one portion and thesuspension was stirred at 50° C. for a further 24 hours. Ethanol (500mL) was charged to the thick reaction mixture and the mixture wasallowed to cool to room temperature. The resulting suspension wasfiltered and the solid washed with ethanol (1 L) and dried in vacuo togive 3-amino-6-bromopyrazine-2-carbohydrazide (98 g, quantitative) as acream solid: ¹H NMR Spectrum; (DMSO-d₆) 4.52 (2H, s), 7.59 (2H, s), 8.30(1H, s), 9.74 (1H, s); Mass Spectrum [M+H]⁺=232.

Pivalic anhydride (165 mL, 815.38 mmol) was added to a stirred mixtureof 3-amino-6-bromopyrazine-2-carbohydrazide (172 g, 741.26 mmol) inacetonitrile (1.8 L) and the mixture was heated at 80° C. for 1 hour.The reaction was left to stir for 16 hours. The required yellow solidmaterial was isolated by filtration. The filtrate was partitionedbetween EtOAc (2 L) and aqueous sodium bicarbonate (2 L). The organiclayer was washed with saturated brine and dried over MgSO₄. The solutionwas filtered and concentrated to give an orange sticky solid which wastriturated with MTBE (250 mL). The insoluble yellow solid was isolatedby filtration and this material was shown to be identical to the firstsolid. The combined solids were dried in the vacuum oven at 50° C. for 3days to afford 3-amino-6-bromo-N′-pivaloylpyrazine-2-carbohydrazide (224g, 96%) as a yellow solid: ¹H NMR Spectrum: (DMSO-d₆) 1.17 (9H, s), 7.62(2H, s), 8.37 (1H, s), 9.42-9.56 (1H, m), 10.09-10.23 (1H, m); MassSpectrum [M+H]⁺=318.

p-Toluenesulfonyl chloride (164 g, 861.60 mmol) was added portion wiseto a suspension of 3-amino-6-bromo-N′-pivaloylpyrazine-2-carbohydrazide(227 g, 718.00 mmol) and N,N-diisopropylethylamine (297 mL, 1795.01mmol) in acetonitrile (2200 mL). The mixture was stirred for 2 hours at70° C. The reaction was left to cool to room temperature overnight. Thereaction mixture was partitioned between ethyl acetate (2 L) and sodiumbicarbonate solution (2 L). The organic layer was washed with saturatedbrine, dried with magnesium sulfate, filtered, and concentrated underreduced pressure. The resulting brown/beige solid was triturated withhot MTBE (1000 mL) and isolated by filtration and dried to afford5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine as a yellowsolid (187 g, 87%). The mother liquors were evaporated to dryness. Thecrude solid was triturated with MTBE (500 mL) filtered and washed with100 mL of MTBE. The resulting solid was air dried overnight to afford asecond crop of5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (36 g,17%): ¹H NMR Spectrum: (DMSO-d₆) 1.43 (9H, s), 7.70 (2H, s), 8.39 (1H,s); Mass Spectrum [M+H]⁺=298.

In an alternative preparation, to3-amino-6-bromo-N′-pivaloylpyrazine-2-carbohydrazide (2301 g, 7.28 mol)in MeCN (10.8 L) was added DIPEA (3.044 L, 17.48 mol) andp-toluenesulfonyl chloride (1665 g, 8.73 mol) portion-wise (˜280 g×6) at50° C. over a period of 30 mins. The reaction temperature was maintainedbetween 65-70° C. by controlling the rate of addition. After theaddition was complete, the reaction mixture was stirred at 70° C. for 1h. The mixture was cooled to room temperature and quenched with 5%NaHCO₃ (aqueous, 24.2 L). The resulting suspension was stirred for 30min then filtered. The product was washed with water (14.8 L), pulleddry and dried at 50° C. for 16 h. The product was dissolved in DCM (12L) and the phases separated. The organic phase was loaded onto a silicapad (6 kg) and the product was eluted with 20% EtOAc/DCM (8×10 L).Concentration of the product containing fractions gave 1987 g (92%yield) with a purity of 99.8% by HPLC.

A stream of nitrogen gas was passed through a solution of5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (89.35 g,239.75 mmol) in DMA (1.2 L) for 20 minutes.Dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine (11.43 g,23.98 mmol), tris(dibenzylideneacetone)dipalladium(0) (5.49 g, 5.99mmol), zinc (1.568 g, 23.98 mmol) and dicyanozinc (16.89 g, 143.85 mmol)were added sequentially to the stirred mixture. The mixture was heatedto 100° C. and stirred for 1 hour. The mixture was cooled andpartitioned between DCM (3 L) and water (1 L). The black mixture wasfiltered through celite and the organic layer was separated. Thesolution was washed with water then brine. The solution was dried withmagnesium sulfate and concentrated under reduced pressure. The residuewas triturated with MTBE and isolated by filtration, washing with MTBE.The filter cake was dried in vacuo to afford5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile(55.7 g, 95%) as a pale orange solid: ¹H NMR Spectrum: (DMSO-d₆) 1.46(9H, s), 6.02 (1H, s), 8.38 (2H, s); Mass Spectrum [M−H]⁻=242.

The product may be slurried in heptanes then filtered and dried as analternative to trituration with MTBE.

Hydrazine hydrate (82 mL, 1.69 mol) was added to5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile (55g, 225.18 mmol) in IPA (200 mL) and the mixture was heated at 50° C.under nitrogen for 16 hours. The mixture was cooled in an ice bath. Theresulting precipitate was collected by filtration, washed with IPA anddiethyl ether and dried to a constant weight to afford(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide(49.2 g, 79%) as a yellow solid: ¹H NMR Spectrum: (DMSO-d₆) 1.45 (9H,s), 5.26 (2H, s), 5.58 (2H, s), 7.56 (2H, s), 8.75 (1H, s); MassSpectrum [M+H]⁺=277.

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (74.3 g, 195.44 mmol) was added to a solution ofN-Boc-isonipecotic acid (41.1 g, 179.15 mmol) and 4-methylmorpholine(35.9 mL, 325.74 mmol) in DMA (800 mL). The mixture was stirred for 10minutes then(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide(45 g, 162.87 mmol) was added to the solution in one portion (exothermobserved from 22° C. to 27° C.). After a few minutes the productcrystallised from the reaction mixture. The reaction mixture was removedfrom the vessel and filtered through a sinter. Additional DMA was addedto wash product from the sides of the vessel (150 mL) and this waspoured onto the filter cake. Isopropanol (600 mL) was added to thevessel and the remainder of the product in the vessel was suspended inthis solvent using vigorous agitation. The isopropanol suspension wasused to wash the filter cake once the DMA had been removed by suction.The filter cake was sucked dry then washed with MTBE and sucked dry onceagain. The filter cake was dried in vacuo to afford (Z)-tert-butyl4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)methylene)hydrazinecarbonyl)piperidine-1-carboxylate(76 g, 95%) as a yellow solid:

¹H NMR Spectrum: (DMSO-d₆) 1.40 (9H, s), 1.46 (9H, s), 1.63-1.9 (2H, m),2.33-2.6 (2H, m, obscured by DMSO signal), 2.63-3.03 (2H, m), 3.18-3.48(4H, m, obscured by water signal), 3.88-4.11 (2H, m), 6.43 (2H, s), 7.76(2H, br), 8.84 (0.5H, s), 8.87 (0.5H, s), 9.85 (1H, s); Mass Spectrum[M+H]⁺=488

In an alternative preparation, the N-Boc-isonipecotic acid may be madein situ as follows: Isonipecotic acid (858 g, 3.74 mol) was dissolved inDMA (25.3 L) and 4-methylmorpholine (393 mL, 3.74 mol) added. Stirredfor 5 mins and isobutyl chloroformate (489 mL, 3.74 mol) added. Thereaction mixture was stirred at 25° C. for 2 h and cooled to 15° C.before(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide(940 g, 3.4 mol) was added portionwise over 10 mins. The reactionmixture was stirred for 1-2 h at 15° C. Water (20.5 L) was addedportionwise over 1 h and stirred for a further 1 h before beingfiltered. The filtercake was then washed with water (4×4 L) and pulleddry on the filter before being dried in a vacuum oven at 50° C. untildry to give the desired product.

Acetic acid (200 mL) was added to dioxane (500 mL) in a 3 L fixed doublejacketed vessel and the solution was heated to 70° C. under nitrogen.(Z)-tert-butyl4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)methylene)hydrazinecarbonyl)-piperidine-1-carboxylate(74.5 g, 152.80 mmol) was added portionwise to the warm mixture. After10 minutes the temperature was increased to 100° C. (slight reflux). Thereaction mixture was stirred at 100° C. for 1.5 hours (suspension) thenheld at 80° C. overnight (solution formed after overnight hold). Theresulting solution was concentrated under reduced pressure, then dilutedwith toluene, evaporated to dryness, taken up with toluene andconcentrated again. The residual oil was mixed with some ethyl acetateand concentrated to dryness. A solid crystallised from solution whichwas triturated with MTBE (200 mL) and isolated by filtration. The filtercake was washed with water and MTBE to afford tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(50 g, 70%) as a grey solid.

The filtrate was concentrated under reduced pressure to give a yellowsolid. This material was triturated with MTBE and filtered. The filtercake was washed with ethyl acetate and then MTBE to give a second cropas a pale yellow solid (4.93 g, 7%). This material was identical to thefirst crop: ¹H NMR Spectrum: (DMSO-d₆) 1.17 (9H, s), 1.22 (9H, s),1.29-1.47 (2H, m), 1.67-1.78 (2H, m), 2.57-2.87 (3H, m), 3.57-3.92 (2H,m), 7.56 (2H, br), 8.56 (1H, s), 13.47 (2H, br s); Mass Spectrum[M+H]+=470.

1,8-Diazabicyclo[5.4.0]undec-7-ene (19.87 mL, 132.90 mmol) was added toa suspension of tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(48 g, 102.23 mmol) in 2-methylTHF (300 mL). A dark solution wasobtained after 5 minutes which was treated with charcoal and filteredthrough a celite pad, washing the charcoal and charcoal with additional2-methylTHF (100 mL). The filtrate was stirred with an air stirrer at−5° C. in a 3 L jacketed fixed vessel under an atmosphere of nitrogen.2-methylTHF (100 mL) was added to help stir the yellow suspension.Iodomethane (7.96 mL, 127.78 mmol) was added dropwise over 15 minutes.The mixture was stirred for 2 hours and the reaction mixture was warmedto room temperature. After 16 hours, additional iodomethane (6 mL) andDBU (20 mL) was added and stirring was continued for 16 hours. Themixture was poured into water and stirred for 5 minutes. The insolublematerial was isolated as a beige solid and dried in vacuo to affordtert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(24.77 g, 50.1%). The mother liquors were concentrated under reducedpressure and the residue was purified by flash column chromatography onsilica using MTBE as the eluant. A second crop of the desired product(13.04 g, 26%), was thus obtained as a yellow solid: ¹H NMR Spectrum:(DMSO-d₆) 1.47 (9H, s), 1.51 (9H, s), 1.57-1.76 (2H, m), 1.94-2.1 (2H,m), 2.87-3.09 (3H, m), 3.9-4.08 (2H, m), 4.26 (3H, s), 7.97 (2H, br, s),8.92 (1H, s); Mass Spectrum [M+H]⁺=484

tert-Butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(36.81 g, 76.12 mmol) was added to a solution of 2,2,2-trifluoroaceticacid (100 mL, 1305.87 mmol) in DCM (100 mL). The mixture was stirred for3 hours at room temperature. The mixture was concentrated under reducedpressure. The residue was dissolved in DCM (1.5 L) and added tovigorously stirred concentrated ammonia (150 mL) in water (400 mL). Theaqueous was washed with DCM (400 mL) and the combined organic solutionswere dried with magnesium sulfate, filtered and concentrated to drynessto afford3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(30.0 g, 103%) as a yellow solid:

¹H NMR Spectrum: (DMSO-d₆) 1.44 (9H, s), 1.54-1.69 (2H, m), 1.8-1.92(2H, m), 2.53-2.63 (2H, m), 2.68-2.83 (1H, m), 2.93-3.05 (2H, m), 4.19(3H, s), 7.89 (2H, br), 8.85 (1H, s); Mass Spectrum [M+H]⁺=384.

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (30.4 g, 80.04 mmol), was added portionwise to astirred solution of 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (12.67g, 72.76 mmol) and N-ethyl-N-isopropylpropan-2-amine (25.3 mL, 145.52mmol) dissolved in acetonitrile (200 mL) at 25° C. The resultingsolution was stirred at 25° C. for 20 minutes then3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(30 g, 72.76 mmol) was added portionwise, washing the last portion intothe mixture as a slurry in acetonitrile (100 mL). After stirring for 1hour the precipitate was collected by filtration, washed withacetonitrile and drying in vacuo to afford1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(35.0 g, 89%) as a beige solid. The filtrate was diluted with DCM (600mL), washed with water, dried over magnesium sulfate and concentrated.The residue was purified by flash chromatography on silica gel elutingwith a gradient of 2 to 2.5% 7N ammonia in methanol withdichloromethane. A second crop of product (3.31 g, 6.13 mmol, 8.43%) wasalso obtained as a cream solid. Both samples were combined to give abeige solid: ¹H NMR Spectrum: (DMSO-d₆) 1.44 (9H, s), 1.52-1.79 (4H, m),1.88-2.04 (2H, m), 2.53-2.73 (2H, m), 2.73-2.87 (1H, m), 2.91-3.05 (1H,m), 3.13-3.24 (1H, m), 3.37-3.47 (1H, m), 3.53-3.65 (1H, m), 3.7-3.8(1H, m), 3.81-3.89 (1H, m), 3.89-3.99 (1H, m), 4.20 (3H, s), 4.29-4.4(1H, m), 4.54-4.61 (1H, m), 7.60-8.20 (2H, br), 8.85 (1H, s); MassSpectrum [M+H]⁺=540.

Example 31-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

Pyridine 4-methylbenzenesulfonate (11.62 g, 46.24 mmol) was added to asuspension of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(128 g, 231.19 mmol) in methanol (1 L) under nitrogen. The mixture wasstirred at 60° C. for 1.5 hours. The mixture was soluble after 5minutes. The mixture was held at 50° C. overnight during which time aprecipitate formed. The solid material was isolated by filtration andwashed with water and acetonitrile. This material still contained minorimpurities from the previous stage and required further purification.The material was dissolved in dichloromethane and purified by flashchromatography on silica gel (0% methanol/DCM to 10% methanol/DCM). Thedesired product,1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(Example 3) (92 g, 85%), was thus isolated as a cream solid (Form A): ¹HNMR Spectrum: (DMSO-d₆) 1.4-1.51 (12H, m), 1.51-1.78 (2H, m), 1.89-2.05(2H, m), 2.72-2.86 (1H, m), 2.91-3.05 (1H, m), 3.12-3.24 (1H, m), 3.64(2H, q), 3.83-4.01 (1H, m), 4.29-4.41 (1H, m), 4.47 (1H, t), 4.58 (2H,q), 8.26 (2H, s), 8.85 (1H, s); Mass Spectrum [M+H]⁺=470.

1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(Example 3.1) was prepared as follows:

1,8-Diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) was added to asuspension of tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(150 g, 319.46 mmol) in 2-methylTHF (1.2 L). Iodoethane (46 mL, 575.03mmol) was added and the mixture was stirred for 16 hours at 35° C.Further iodoethane (46 mL, 575.03 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) were added andstirring was continued for 24 hours at 35° C. The mixture was pouredinto water and the insoluble material was isolated by filtration, washedwith water and MTBE and dried in vacuo to afford tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(116 g, 73.0%) as a yellow solid. The filtrate was extracted with DCMand the organic solution was dried with magnesium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica using gradient elution (30% MTBE/heptaneto 100% MTBE). A second crop of the desired product (12 g, 24.12 mmol,7.55%), was thus isolated as a yellow solid which was later combinedwith the first crop: ¹H NMR Spectrum: (DMSO-d₆) 1.41 (9H, s), 1.44 (9H,s), 1.48 (3H, t), 1.52-1.69 (2H, m), 1.87-2.04 (2H, m), 2.79-3.03 (3H,m), 3.86-4.03 (2H, m), 4.59 (2H, q), 7.89 (2H, s), 8.85 (1H, s); MassSpectrum [M+H]⁺=498.

THF may also be a suitable solvent for the above reaction.

TFA (400 mL) was added portionwise to a solution of tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(126 g, 253.22 mmol) in DCM (400 mL). The mixture was stirred for 16hours at room temperature. The mixture was concentrated under reducedpressure, dissolved in DCM (1 L) and added slowly to a vigorouslystirred solution of concentrated aqueous ammonia (500 mL) in water at 0°C. The organic solution was separated from the aqueous and concentratedunder reduced pressure* to afford3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(101 g, 100%) as a yellow solid: ¹H NMR Spectrum: (DMSO-d₆) 1.4-1.52(12H, m), 1.57-1.73 (2H, m), 1.83-1.93 (2H, m), 2.57-2.7 (2H, m),2.71-2.84 (1H, m), 2.96-3.09 (2H, m), 4.58 (2H, q), 8.06 (2H, s), 8.84(1H, s); Mass Spectrum [M+H]⁺=398. *In another experiment on a similarscale (approximately 170 g of starting material) the following isolationprocedure was utilised: The layers were separated and the top layer(emulsion with a solid) was filtered. The solid was washed with DCM (0.5L) and the filtrate was transferred to a separating funnel. The layerswere separated and the aqueous layer was extracted with DCM (0.5 L). Theorganic layers were dried over MgSO₄, filtered and concentrated. Theproduct was dried at 50° C. overnight (81.75 g). The solid from theextraction was slurried in water (200 mL) for 30 min at room temperatureand filtered off. The product was dried at 50° C. in vacuo (61.8 g).

A further variation is as follows;

A suspension of tert-butyl4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate(3009.5 g, 6.05 mol) in DCM (9 L) was cooled to 5-10° C. under N₂. TFA(9 L) was added portionwise to the suspension whilst maintaining thetemperature <30° C. The reaction mixture was stirred at room temperaturefor 1 h. The mixture was concentrated, the resulting residue wasdissolved in water (30 L) and added slowly to a 35% aqueous ammoniasolution (12 L) at 0-5° C. The suspension was stirred for 30 min thenthe product was filtered off and washed with water (2×6 L). The productwas dried at 50° C. in vacuo for 2 days (2496 g).

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 106 g, 279.51 mmol), was added portionwise toa stirred solution of 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid(44.3 g, 254.10 mmol) and N-ethyl-N-isopropylpropan-2-amine (89 mL,508.21 mmol) dissolved in acetonitrile (600 mL) at 25° C. The resultingsolution was stirred at 25° C. for 20 mn then3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(101 g, 254.10 mmol) was added portionwise washing the last portion intothe mixture as a slurry in acetonitrile (300 mL). After stirring for 1hour the precipitate was collected by filtration, washing withacetonitrile and drying in vacuo to afford1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(128 g, 91%) as a beige solid. The filtrate was diluted with DCM (600ml), washed with water, dried over magnesium sulfate and concentrated.The residue was purified by flash chromatography on silica gel elutingwith a gradient of 2 to 2.5% 7N ammonia in methanol withdichloromethane. A second crop of the desired product (40 g, 72.2 mmol,28.4%) was obtained as a cream solid which was combined with the firstcrop:

¹H NMR Spectrum: (DMSO-d₆) 1.29-1.48 (16H, m), 1.48-1.75 (4H, m),1.83-1.99 (2H, m), 2.48-2.68 (2H, m), 2.68-2.79 (1H, m), 2.87-2.99 (1H,m), 3.07-3.19 (1H, m), 3.32-3.42 (1H, m), 3.47-3.6 (1H, m), 3.64-3.75(1H, m), 3.75-3.84 (1H, m), 3.84-3.95 (1H, m), 4.24-4.39 (1H, m),4.47-4.6 (3H, m), 7.84 (2H, s), 8.79 (1H, s): Mass Spectrum [M+Na]⁺=577.

Alternative Preparation:

To a solution of 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (48.80 g0.2774 mol) and N-ethyl-N-isopropylpropan-2-amine (86.96 mL, 0.4993 mol)in THF (552 mL) was addedO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (115.73 g, 0.3051 mol) portionwise at RT undernitrogen. The resulting mixture was stirred for 20 min then3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(122.5 g (110.25 g active), 0.2774 mol) was added portionwise over 1 h.After 3.5 h, the mixture was concentrated and the residue was slurriedin MeCN (275 mL) for 15 min at room temperature. The product wasfiltered off, washed with MeCN (3×110 mL) and dried overnight at 50° C.in vacuo. This gave1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(131.9 g, 96%).

In a further alternative preparation, HBTU(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)in THF may be used as coupling agent instead of HATU.

Alternative Preparation of Example 3

To a suspension of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one(131.9 g, 0.2382 mol) in methanol (1045 mL) was added pyridiniump-toluenesulfonate (11.97 g, 47.7 mmol) under N2. The reaction mixturewas stirred at 60° C. for 5.5 h then at 50° C. overnight. The reactionmixture was cooled to 0° C. and the solid was filtered off. The productwas slurried in water (250 mL) for 20 min at room temperature, filteredoff, washed with water (3×40 mL) and dried at 50° C. in vacuo. This gave1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(21.4 g) as Form A (see below).

The methanol liquors were concentrated and the resulting solid wasslurried in water (0.6 L) for 20 min at room temperature. The solid wasisolated by filtration and washed with water (3×100 mL). The filter cakewas slurried for a second time in water (0.5 L) for a further 20minutes. The product was isolated by filtration, washed with water (100mL) and dried at 50° C. in vacuo. This gave 81.9 g1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(81.9 g) as Form A.

Both crops were combined (103.3 g), seeded with Form B (16.68 g) andslurried in MeCN (826 mL) at room temperature overnight. This gave 117.4g of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-oneas a pale yellow solid (117.4 g), Form B (see below). This material wasfurther purified by slurrying in heptane (7.5 rel vols) for 1 hour. Themixture was filtered, pulled dry on the filter, and dried at 50° C. in avacuum oven overnight to afford1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(102.5 g) as Form B.

Form B may also be made by slurrying Form A in MeCN without seeding.

Form A or B may also be converted to Form C as follows:

A suspension of1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one(eg Form B made by the processes outlined above) in IPA (12 vol) washeated at reflux until the solid dissolved. The solution was hotfiltered then cooled to room temperature. This gave1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-oneas a pale yellow solid (99.3 g, 97%) as Form C.

Form C may also be converted to Form B as follows:

In a 10 L flange flask, Form C (377.8 g portion 1) in MIBK (7900 mL) washeated to 110-115° C. to give a solution. The solution was allowed tocool to 97-103° C. and immediately polish filtered into a 50 L vesselcontaining a seed of Form B (0.8 g) in acetonitrile (8220 mL) stirringat −15° C. During the addition the temperature in the 50 L vessel wasmaintained between −15 and 25° C. by means of jacket cooling. Threefurther portions of the compound dissolved in MIBK were added by asimilar method. To the resulting slurry was added a seed of form B (0.8g) and the mixture was then stirred at 10-20° C. overnight. In-processanalysis confirmed the desired form (Form B) with no Form C or amorphousvisible. The mixture was filtered and washed with acetonitrile (3340mL). The solid was oven dried for 2 days (solid was broken up during thedrying to a powder and a mixture of small lumps ˜1 mm to ˜3-4 mm size)until constant weight was obtained.

Yield=1532.8 g (93.5%)

3-(Tetrahydro-2H-pyran-2-yloxy)propanoic acid was prepared as follows:

To a stirred solution of methanol (2.4 L) and concentrated sulfuric acid(44.4 mL, 832.61 mmol) at 0 OC under nitrogen was added, dropwise,beta-propiolactone (175 mL, 2.78 mol). This solution was allowed to stirat room temperature for 2 days. The reaction mixture was cooled to 10 OCbefore adding, portionwise, sodium bicarbonate (145 g, 1.72 mol), theresulting suspension was left to stir at room temperature for 75minutes. This solution was filtered, the filter-cake was washed withmethanol (800 mL). The filtrate was evaporated to an oil which wasredissolved in dichloromethane (1.2 L) and stirred for 60 minutes beforerefiltering. This solution was filtered before evaporating to givemethyl 3-hydroxypropanoate (219 g, 76%) as an oil. ¹H NMR Spectrum:(CDCl₃) 2.50 (2H, t), 3.63 (3H, s), 3.78 (2H, t).

Pyridinium p-toluenesulfonate (7.65 g, 30.45 mmol) was added to a clearsolution of methyl 3-hydroxypropanoate (63.4 g, 609.00 mmol) and3,4-dihydro-2H-pyran (78 mL, 852.60 mmol) in dichloromethane (650 mL) atroom temperature under nitrogen to give a cloudy solution. This wasallowed to stir at room temperature overnight. The reaction mixture waswashed with water (250 mL) and brine (250 mL) before drying (MgSO₄) andevaporating to an oil. This crude product was purified by flash silicachromatography, elution gradient 15 to 30% EtOAc in heptane. Purefractions were evaporated to dryness to afford methyl3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.7 g, 59.0%) as acolourless oil: ¹H NMR Spectrum: (CDCl₃) 1.47 (4H, dddd), 1.55-1.84 (2H,m), 2.55 (2H, t), 3.33-3.53 (1H, m), 3.53-3.7 (4H, m), 3.78 (1H, ddd),3.93 (1H, dt), 4.42-4.72 (1H, m); Mass Spectrum [MH]⁺=189.

Sodium hydroxide (2M, 349 mL, 697.58 mmol) was added to a solution ofmethyl 3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.68 g, 359.58 mmol)in THF (680 mL) at room temperature. The reaction mixture was stirred atroom temperature for 3 hours. The THF was removed in vacuo, the aqueouslayer was then washed with ethyl acetate (260 mL), before cooling to 0°C. and careful acidification to pH 5 by the addition of hydrochloricacid (2M). The product was extracted with ethyl acetate (3×250 mL)before drying (MgSO₄) and evaporation to give3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (57.0 g, 91%) as a clearoil. This material was dissolved in ethyl acetate (750 mL) then washedwith water (3×250 mL) and brine (250 mL) to remove remaining aceticacid. The organic solution was dried (MgSO₄) and evaporated to give3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (45.67 g, 72.9%) as acolourless oil: ¹H NMR Spectrum: 1H NMR (CDCl₃) 1.43-1.67 (4H, m),1.65-1.95 (2H, m), 2.68 (2H, t), 3.48-3.58 (1H, m), 3.73 (1H, dt), 3.88(1H, ddd), 4.02 (1H, dt), 4.59-4.7 (1H, m); Mass Spectrum [M−H]⁻=173.Example 3 as isolated above was a crystalline solid in three differentcrystalline forms, described herein as Forms A, B and C.

The crystal structure of Form A of Example 3 may be characterised byXRPD and DSC.

The methods for carrying out these techniques are as described forExample 1.

Ten X-Ray Powder Diffraction peaks for Example 3 Form A Angle 2- Theta(2θ) Intensity % 4.8 100 10.0 89.2 14.6 81.9 5.2 59.4 19.9 53.6 10.449.3 25.4 48.7 23.6 48.6 24.4 43.9 16.2 36.3

The XRPD for Example 3 Form A is shown in FIG. 3.

DSC analysis of Example 3 Form A shows an initial endotherm with anonset of 27.0° C. and a peak at 63.0° C., further endothermic shifts areseen with onsets and peaks at the following temperatures; 166.5° C. and168.7° C., 172.2° C. and 173.2° C. and a final melt at 174.8° C. and apeak at 175.7° C. (FIG. 4).

Thus DSC analysis shows Example 3 Form A is a solvated material with anonset of desolvation at about 27.0° C. and a peak at about 63.0° C.

The X-ray powder diffraction spectra for Example 3 (Form A) showed thematerial to be crystalline. This material had a desolvation point of28.0° C. (onset).

Example 3 can also exist in an alternative polymorphic form, referred toherein as Form B. Preparation of Form B was described above.

This material had a melting point of 172.5° C. (onset).

In a further aspect of the invention, there is provided a process formaking Form B of Example 3 by slurrying a sample of Example 3 inacetonitrile. In a further aspect of the invention there is provided aprocess for making Form B of Example 3 from a solution of Form C ofExample 3 in MIBK.

Ten X-Ray Powder Diffraction peaks for Example 3 Form B Angle 2- Theta(2θ) Intensity % 5.8 100.0 10.9 59.8 11.5 33.8 25.9 18.2 17.3 15.8 24.014.1 19.1 13.4 12.9 11.7 24.7 11.1 27.2 9.7

The XRPD for Example 3 Form B is shown in FIG. 5.

DSC analysis of Example 3 Form B shows a melting endotherm with an onsetof 172.5° C. and a peak at 174.2° C. (FIG. 6).

Thus DSC analysis shows Example 3 B is a high melting solid with anonset of melting at about 172.5° C. and a peak at about 174.2° C.

Example 3 may also exist in a third crystalline form, referred to hereinas Form C. A process for making Form C material from eg Form B materialwas described above, by crystallisation from isopropyl alcohol (IPA).

Therefore in a further aspect of the invention there is provided aprocess for making Form C of Example 3 by crystallising Example 3 fromIPA.

Example 3 Form C is characterised in providing at least one of thefollowing 2θ values measured using CuKa radiation: 6.9 and 12.3. Example3 Form C is characterised in providing an X-ray powder diffractionpattern, substantially as shown in Figure A. Ten X-Ray powderdiffraction peaks are shown below:

Ten X-Ray Powder Diffraction peaks for Example 3 Form C Angle 2- Theta(2θ) Intensity % 6.9 40.1 12.3 100.0 10.5 23.8 21.0 67.9 24.6 36.1 13.621.4 16.4 19.9 19.6 18.1 20.2 17.5 22.5 18.4

DSC analysis of Example 3 Form C shows a melting endotherm with an onsetof 183.0° C. and a peak at 185.6° C. (Figure B).

Thus DSC analysis shows Example 3 Form C is a high melting solid with anonset of melting at about 183.0° C. and a peak at about 185.6° C.

Details of Techniques Used for Form C analysis

X-Ray Powder Diffraction

Analytical Instrument: Panalytical Cubix.

The X-ray powder diffractogram was determined by mounting a sample ofthe crystalline material on a Panalytical single silicon crystal (SSC)wafer mount and spreading out the sample into a thin layer with the aidof a microscope slide. The sample was spun at 30 revolutions per minute(to improve counting statistics) and irradiated with X-rays generated bya copper long-fine focus tube operated at 45 kV and 40 mA with awavelength of 1.5418 angstroms. The X-ray beam was passed through a 0.04rad soller slit, then an automatic variable divergence slit set at 20 mmand finally a 20 mm beam mask. The reflected radiation was directedthrough a 20 mm antiscatter slit and a 0.04 rad soller slit. The samplewas exposed for 1.905 seconds per 0.0025067° 2-theta increment(continuous scan mode) over the range 2 degrees to 40 degrees 2-theta intheta-theta mode. The instrument was equipped with an X-Celeratordetector. Control and data capture was by means of a Dell Pentium 4HTWorkstation operating with X'Pert Industry software. Persons skilled inthe art of X-ray powder diffraction will realise that the relativeintensity of peaks can be affected by, for example, grains above 30microns in size and non-unitary aspect ratios that may affect analysisof samples. The skilled person will also realise that the position ofreflections can be affected by the precise height at which the samplesits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values.

Differential Scanning Calorimetry

Analytical Instrument: TA Instruments Q1000 DSC.

Typically less than 5 mg of material contained in a standard aluminiumpan fitted with a lid was heated over the temperature range 25° C. to300° C. at a constant heating rate of 10° C. per minute. A purge gasusing nitrogen was used—flow rate 50 ml per minute.

Example 4(3R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one

2-(1H-Benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouroniumtetrafluoroborate (201 mg, 0.63 mmol) was added to a stirred suspensionof3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(200 mg, 0.52 mmol, described in Example 1),N-ethyl-N-isopropylpropan-2-amine (0.273 mL, 1.56 mmol) and(R)-3-hydroxybutanoic acid (65.2 mg, 0.63 mmol) in N,N-dimethylformamide(3 mL). The resulting suspension was stirred at room temperature for 2hours. The resulting mixture was purified by preparative HPLC using aWaters X-Bridge reverse-phase column (C-18, 5 microns silica, 30 mmdiameter, 150 mm length, flow rate of 60 ml/minute) using an isocraticmixture of 31% acetonitrile in water (containing ammonium carbonate (2g/L). The fractions containing the desired compound were evaporated todryness to afford a pale yellow solid. This solid was stirred inacetonitrile (3 mL) at room temperature. The resulting solid wasfiltered, washed with cold acetonitrile and dried to afford the titlecompound (125 mg, 51.0%) as a pale yellow solid.

¹H NMR Spectrum: (CDCl₃) 1.24 (3H, d), 1.52 (9H, s), 1.85 (2H, m), 2.10(2H, m), 2.35 (1H, dd), 2.55 (1H, d), 2.90 (1H, m), 3.05 (1H, m), 3.20(1H, m), 3.90 (1H, m), 4.25 (1H, m), 4.31 (3H, s), 4.6 (1H, m), 9.03(1H, s); Mass Spectrum [M+H]⁺=470.

Example 5(3S)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one

Using a similar procedure as Example 4,3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-aminewas reacted with (S)-3-hydroxybutanoic acid to afford the title compound(167 mg, 68.2%) as a pale yellow solid.

¹H NMR Spectrum: (CDCl₃) 1.24 (3H, d), 1.52 (9H, s), 1.85 (2H, m), 2.10(2H, m), 2.35 (1H, dd), 2.55 (1H, d), 2.90 (1H, m), 3.05 (1H, m), 3.20(1H, m), 3.90 (1H, m), 4.25 (1H, m), 4.31 (3H, s), 4.6 (1H, m), 9.03(1H, s); Mass Spectrum [M+H]⁺=470.

Example 6(2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-2-methyl-propan-1-one

Using a similar procedure as Example 4,3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-aminewas reacted with (R)-3-hydroxy-2-methylpropanoic acid to afford thetitle compound (87 mg, 47.4%) as a pale yellow solid.

¹H NMR Spectrum: (CDCl₃) 1.55 (9H, s), 1.61 (3H, s br), 1.8-2.0 (2H, m),2.10-2.25 (2H, m), 2.90 (2H, m), 3.10 (1H, m), 3.3 (2H, m), 3.77 (2H,m), 4.33 (3H, s), 4.6 (1H, m), 9.05 (1H, s); Mass Spectrum [M+H]+=470.

Example 71-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-2-hydroxy-2-methyl-propan-1-one

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg,0.52 mmol) was added in one portion to 2-hydroxy-2-methylpropanoic acid(38.0 mg, 0.37 mmol),3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(100 mg, 0.26 mmol) and 2-hydroxy-pyridine N-oxide (57.9 mg, 0.52 mmol)dissolved in NMP (1.2 mL) under argon. The resulting solution wasstirred at 25° C. for 3 hours. Pyridine (100 μL, 1.24 mmol) was addedand the mixture was stirred for 18 hours. Additional 2-hydroxypyridine1-oxide (57.9 mg, 0.52 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg,0.52 mmol) was added. The mixture was then heated up to 70° C. for 48hours, more 2-hydroxy-2-methylpropanoic acid (15 mg, 0.14 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50.0 mg,0.26 mmol) and 2-hydroxypyridine 1-oxide (25.0 mg, 0.23 mmol) were addedand the mixture was then kept to 70° C. for 8 hours. The solution waspurified by preparative HPLC using a Waters X-Bridge reverse-phasecolumn (C-18, 5 microns silica, 19 mm diameter, 100 mm length, flow rateof 40 ml/minute) and decreasingly polar mixtures of water (containing0.2% ammonium carbonate) and acetonitrile as eluent to afford the titlecompound (71 mg, 58%) as a pale yellow solid.

¹H NMR Spectrum: (CDCl₃) 1.55 (15H, s br), 1.90 (2H, m), 2.15 (2H, m),3.05-3.3 (4H, m), 4.32 (3H, s), 4.6 (1H, m), 9.03 (1H, s); Mass Spectrum[M+H]⁺=470.

Example 83-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-oxo-propanoicacid

Ethyl 3-chloro-3-oxopropanoate (0.037 mL, 0.29 mmol) was added dropwiseto a stirred solution of3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(100 mg, 0.26 mmol) and triethylamine (0.047 mL, 0.34 mmol) dissolved inCH₂Cl₂ (1.5 mL) over a period of 2 minutes at 0 OC under nitrogen. Themixture was stirred at 0 OC for 10 minutes then allowed to warm to roomtemperature and stirred for 1 hour. The mixture was evaporated,dissolved in DMF; a white solid was filtered off and the filtrate waspurified by preparative HPLC using a Waters X-Terra reverse-phase columneluting with a mixture of water (containing 0.2% ammonium carbonate) andacetonitrile to afford ethyl3-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-oxopropanoate(80 mg, 61.7%) as a yellow solid. This material was suspended in THF (2mL). 2N Sodium hydroxide (0.235 mL, 0.47 mmol) and water (0.5 ml) wereadded. The mixture was stirred at room temperature overnight. 2NHydrochloric acid (230 μL) was added to the mixture. The solvents wereevaporated. The residue was diluted with CH₂Cl₂ (30 mL) and water (5mL). The organic phase was washed with brine and dried over MgSO₄. Thesolvents were evaporated. The resulting foam was triturated in ether.The resulting yellow solid was filtered, dried, triturated inacetonitrile (3 mL). The yellow solid was collected by filtration, driedat 40° C. to afford the title compound (50 mg, 68%) as a yellow solid.

¹H NMR Spectrum: (DMSO-d₆) 1.46 (9H, s), 1.58 (1H, m), 1.74 (1H, m),1.98 (2H, m), 2.84 (1H, m), 3.0 (1H, m), 3.21 (1H, m), 3.46 (2H, m),3.83 (1H, m), 4.22 (3H, s), 4.34 (1H, m), 7.8-8.2 (1H, m), 8.87 (1H, s);Mass Spectrum [M+H]⁺=470.

Example 93-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-oxo-propanoicacid

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (474 mg, 1.25 mmol) was added over 30 seconds inportions to a stirred solution of 3-ethoxy-3-oxopropanoic acid (150 mg,1.13 mmol), N-ethyl-N-isopropylpropan-2-amine (0.394 mL, 2.26 mmol) and3-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(450 mg, 1.13 mmol) dissolved in DMF (20 mL) at 50° C. The resultingsolution was sampled after 1 min (complete reaction) and immediatelyallowed to cool to ambient temperature. The reaction mixture wasconcentrated and diluted with EtOAc (100 mL), and washed sequentiallywith water (20 mL) and saturated brine (20 mL). The organic layer wasdried over MgSO₄, filtered and evaporated to afford crude ethyl3-(4-(5-(5-amino-6-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-oxopropanoate(850 mg).

Some of that material (780 mg) was dissolved in THF (20 ml). To thissolution was added 2N aqueous sodium hydroxide (2.3 ml, 4.57 mmol) andwater (5 ml) followed by methanol (5 ml) to give a clear solution. Themixture was stirred at room temperature for 3 hours. The THF wasevaporated. The aqueous layer was acidified to pH3 with 2N aqueoushydrochloric acid (2.5 ml). Dichloromethane (50 ml) was added and theorganic phase extracted. The organic phase was washed with brine (10 ml)and dried over MgSO₄. The solvents were evaporated. The resulting gumwas purified by preparative HPLC (Waters X-Bridge Prep C18 OBD column,5μ silica, 50 mm diameter, 100 mm length), using decreasingly polarmixtures of water (containing 1% ammonia) and acetonitrile as eluents.Fractions containing the desired compound were evaporated to dryness toafford pure ammonium salt. This was solubilised in water and acidifiedto pH3 with 2N hydrochloric acid (˜0.3 ml). Dichloromethane (50 mL) wasadded and the organic phase separated, washed with brine (5 ml) anddried over MgSO₄. After filtration the resulting solution was evaporatedto dryness and the residue was triturated with diethyl ether (5 mL) andfiltered to afford3-(4-(5-(5-amino-6-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-oxopropanoicacid (195 mg, 26.5%) as a cream solid.

¹H NMR Spectrum: (DMSO-d₆) 1.45 (9H, s), 1.48 (3H, m), 1.55-1.62 (1H,m), 1.70-1.80 (1H, m), 1.95-2.05 (2H, m), 2.80-2.90 (1H, m), 2.95-3.05(1H, m), 3.15-3.25 (1H, m), 3.45 (2H, s), 3.78-3.85 (1H, m), 4.30-4.40(1H, m), 4.55-4.65 (2H, m), 7.80-8.00 (2H, br s), 8.88 (1H, s), 12.60(1H, s); Mass Spectrum [M+H]⁺=484

Example 10(2S)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-2,3-dihydroxy-propan-1-one

To a mixture of3-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine(257 mg, 0.50 mmol, TFA salt), potassium(S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (101 mg, 0.55 mmol) andEDCI (105 mg, 0.55 mmol) in DCM (5 mL) were added1-hydroxy-1H-benzotriazol hydrate (85 mg, 0.56 mmol) and DIPEA (194 mg,1.50 mmol). The mixture was stirred for 16 hours at room temperature.Water was added to the mixture and the mixture was extracted with DCM.The organic layers were washed with brine and dried over Na₂SO₄,filtered and concentrated to give(S)-(4-(5-(5-amino-6-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)(2,2-dimethyl-1,3-dioxolan-4-yl)methanone(320 mg). Mass Spectrum [M+H]⁺=526. To a mixture of(S)-(4-(5-(5-amino-6-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)(2,2-dimethyl-1,3-dioxolan-4-yl)methanone(320 mg) in DCM (10 mL) at r.t was added dropwise TFA (1.6 ml, 20.77mmol). The mixture was stirred for 16 h at r.t, concentrated andpurified by preparative HPLC (Waters XBridge Prep C18 OBD column, 5μsilica, 19 mm diameter, 100 mm length) using decreasingly polar mixturesof water (containing 0.1% NH₃) and MeCN as the eluant. The fractionscontaining the desired compound were evaporated to dryness to afford thetitle compound (142 mg, 48%) as a white solid. 1H NMR Spectrum (400 Hz,DMSO-d₆, 30° C.): 1.45 (12H, m), 1.56 (1H, m), 1.70 (1H, m), 1.98 (2H,m), 2.85 (1H, m), 3.00 (1H, m), 3.20 (1H, m), 3.45 (1H, s), 3.55 (1H,s), 4.05 (1H, m), 4.35 (2H, m), 4.60 (2H, m), 4.70 (1H, m), 4.85 (1H,m), 7.90 (2H, m), 8.85 (1H, s); Mass Spectrum [M+H]⁺=486.

Example 11(2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-2,3-dihydroxy-propan-1-one

3-(5-(Tert-butyl)-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-aminewas reacted with potassium (R)-2,2-dimethyl-1,3-dioxolane-4-carboxylate,using a similar procedure than described in Example 10 to provide thetitle compound (0.145 g, 40%) as a solid. ¹H NMR Spectrum (400 Hz,DMSO-d₆, 30° C.): 1.45 (12H, m), 1.60 (2H, m), 1.98 (2H, m), 2.85 (1H,m), 3.00 (1H, m), 3.17 (1H, m), 3.45 (1H, s), 3.55 (1H, s), 4.05 (1H,m), 4.35 (2H, m), 4.60 (2H, m), 4.70 (1H, m), 4.85 (1H, m), 7.90 (2H,m), 8.85 (1H, s); Mass Spectrum [M+H]⁺=486.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an X-Ray powder Diffraction Pattern for Example 1 Form A.

FIG. 2 shows a DSC Thermogram for Example 1 Form A.

FIG. 3 shows an X-Ray powder Diffraction Pattern for Example 3 Form A.

FIG. 4 shows a DSC Thermogram for Example 3 Form A.

FIG. 5 shows an X-Ray powder Diffraction Pattern for Example 3 Form B.

FIG. 6 shows a DSC Thermogram for Example 3 Form B.

FIG. 7 shows an X-Ray powder Diffraction Pattern for Example 3 Form C.

FIG. 8 shows a DSC Thermogram for Example 3 Form C.

FIG. 9 shows Tumour Growth Inhibition by Example 3 in Combination withAKT inhibitor (AZD5363)—sequential administration

FIG. 10 shows Tumour Growth Inhibition by Example 3 in Combination withAKT inhibitor (AZD5363), co-administration

FIG. 11 shows Tumour Growth Inhibition by Example 3 in Combination withPARP inhibitor (Olaparib) in BT474 xenograft model

FIG. 12 shows Tumour Growth Inhibition by Example 3 in Combination withPARP inhibitor (Olaparib) MCF7 xenograft model

FIG. 13 shows Tumour Growth Inhibition by Example 3 in Combination with(AZD8186)

1. A compound of the Formula (I)

wherein R¹ is methyl or ethyl; and R² is (C2-3)alkyl substituted byhydroxy; or a pharmaceutically-acceptable salt thereof.
 2. A compound asclaimed in claim 1, or a pharmaceutically-acceptable salt thereof,wherein R² is selected from groups (i) to (xi):


3. A compound as claimed in claim 1, or a pharmaceutically-acceptablesalt thereof wherein R² is selected from groups (i) to (vi) as definedin claim
 2. 4. A compound as claimed in claim 1, or apharmaceutically-acceptable salt thereof wherein R2 is group (i) asdefined in claim
 2. 5. A compound as claimed in claim 1, or apharmaceutically-acceptable salt thereof wherein R1 is methyl.
 6. Acompound as claimed in claim 1, or a pharmaceutically-acceptable saltthereof wherein R1 is ethyl.
 7. A compound as claimed in claim 1 or apharmaceutically-acceptable salt thereof, which compound is selectedfrom:1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one;1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one;(3R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one;(3S)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one;(2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-2-methyl-propan-1-one;1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-2-hydroxy-2-methyl-propan-1-one.8. A compound as claimed in claim 1, or a pharmaceutically-acceptablesalt thereof in a crystalline form.
 9. (canceled)
 10. (canceled)
 11. Acombination suitable for use in the treatment of cancer comprising acompound of Formula (I) as claimed in claim 1 or a pharmaceuticallyacceptable salt thereof and another anti-tumour agent.
 12. Apharmaceutical composition comprising a compound of Formula (I) or apharmaceutically-acceptable salt thereof, as claimed in claim 1, and apharmaceutically-acceptable diluent or carrier.
 13. A method forselecting a patient for treatment with a compound of Formula (I) asclaimed in claim 1, the method comprising providing a sample from apatient containing tumour-derived DNA or tumour cells; determiningwhether the PIK3CA gene in the patient's tumour cells or tumour-derivedDNA are wild type or mutant; and selecting a patient for treatment witha compound of Formula (I) based thereon.
 14. A method of treating apatient suffering from cancer comprising: providing a tumour cellcontaining sample from a patient; determining whether the PIK3CA gene inthe patient's tumour cells are wild type or mutant; and administering tothe patient an effective amount of a compound of Formula (I) as claimedin claim 1 if the tumour cells possess a mutant PIK3CA gene.
 15. Acompound of Formula (I) as claimed in claim 1 for treating cancers withtumour cells identified as harbouring a mutant PIK3CA gene.
 16. Acrystalline form of1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one,or a pharmaceutically acceptable salt thereof.
 17. The crystalline formas claimed in claim 16, which is1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-oneForm A.
 18. The crystalline form as claimed in claim 16, which is1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-oneForm B.
 19. The crystalline form as claimed in claim 16, which is1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-oneForm C.
 20. A method for the prevention or treatment of cancer in a warmblooded animal, such as man, in need of such treatment which comprisesadministering to said animal an effective amount of a compound of theFormula (I) as claimed in claim 1, or a pharmaceutically-acceptable saltthereof.
 21. The method as claimed in claim 20, wherein the compound ofFormula (I) is1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one,or a pharmaceutically-acceptable salt thereof.
 22. The method as claimedin claim 20, wherein the cancer is breast cancer.
 23. The combination asclaimed in claim 11, wherein the compound of Formula (I) is1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one,or a pharmaceutically-acceptable salt thereof.
 24. The combination asclaimed in claim 11, wherein the additional anti-tumour agent isselected from an mTOR inhibitor, a PI3K-β inhibitor, an inhibitor of AKTkinase or olaparib.